Back to Multiple platform build/check report for BioC 3.22:   simplified   long
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This page was generated on 2025-08-07 12:05 -0400 (Thu, 07 Aug 2025).

HostnameOSArch (*)R versionInstalled pkgs
nebbiolo2Linux (Ubuntu 24.04.2 LTS)x86_644.5.1 (2025-06-13) -- "Great Square Root" 4815
palomino8Windows Server 2022 Datacenterx644.5.1 (2025-06-13 ucrt) -- "Great Square Root" 4550
lconwaymacOS 12.7.1 Montereyx86_644.5.1 (2025-06-13) -- "Great Square Root" 4592
kjohnson3macOS 13.7.1 Venturaarm644.5.1 Patched (2025-06-14 r88325) -- "Great Square Root" 4534
Click on any hostname to see more info about the system (e.g. compilers)      (*) as reported by 'uname -p', except on Windows and Mac OS X

Package 728/2315HostnameOS / ArchINSTALLBUILDCHECKBUILD BIN
FLAMES 2.3.4  (landing page)
Changqing Wang
Snapshot Date: 2025-08-06 14:04 -0400 (Wed, 06 Aug 2025)
git_url: https://git.bioconductor.org/packages/FLAMES
git_branch: devel
git_last_commit: af7c6a6
git_last_commit_date: 2025-07-22 19:46:17 -0400 (Tue, 22 Jul 2025)
nebbiolo2Linux (Ubuntu 24.04.2 LTS) / x86_64  OK    OK    OK  UNNEEDED, same version is already published
palomino8Windows Server 2022 Datacenter / x64... NOT SUPPORTED ...
lconwaymacOS 12.7.1 Monterey / x86_64  OK    OK    OK    OK  UNNEEDED, same version is already published
kjohnson3macOS 13.7.1 Ventura / arm64  OK    OK    OK    OK  UNNEEDED, same version is already published


CHECK results for FLAMES on nebbiolo2

To the developers/maintainers of the FLAMES package:
- Allow up to 24 hours (and sometimes 48 hours) for your latest push to git@git.bioconductor.org:packages/FLAMES.git to reflect on this report. See Troubleshooting Build Report for more information.
- Use the following Renviron settings to reproduce errors and warnings.
- If 'R CMD check' started to fail recently on the Linux builder(s) over a missing dependency, add the missing dependency to 'Suggests:' in your DESCRIPTION file. See Renviron.bioc for more information.

raw results


Summary

Package: FLAMES
Version: 2.3.4
Command: /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.4.tar.gz
StartedAt: 2025-08-06 22:33:01 -0400 (Wed, 06 Aug 2025)
EndedAt: 2025-08-06 22:57:26 -0400 (Wed, 06 Aug 2025)
EllapsedTime: 1464.6 seconds
RetCode: 0
Status:   OK  
CheckDir: FLAMES.Rcheck
Warnings: 0

Command output

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.4.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 (2025-06-13)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.2 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.3.4’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 50 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... OK
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  5.9Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for ‘new’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_barcode: no visible binding for global variable ‘Sample’
find_barcode: no visible binding for global variable ‘Outfile’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq
  Outfile Reads Sample Type UMI UMI_count adj.p.value allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file everything expect_cell_number
  expr fastq filter_res genome_bam head imageX imageY in_tissue input j
  length_bin max_length min_length minimap2 multi-matching reads
  mutation_index n_reads na.omit name new nucleotide outdir output
  p.value packageVersion pct pos read1_with_adapter read_counts ref
  samtools scale_alpha_continuous scale_colour_gradient single match
  reads starts_with test threads total total reads total_counts
  tr_length transcript transcriptome_assembly transcriptome_bam
  undemultiplexted reads unzip value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

Compiled code should not call entry points which might terminate R nor
write to stdout/stderr instead of to the console, nor use Fortran I/O
nor system RNGs nor [v]sprintf. The detected symbols are linked into
the code but might come from libraries and not actually be called.

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
blaze                         4.826 18.276  13.471
plot_isoform_reduced_dim     22.626  0.300  22.926
find_variants                20.400  0.302  19.929
sc_long_multisample_pipeline 13.118  5.924  12.703
bulk_long_pipeline            2.789 13.152   2.998
MultiSampleSCPipeline        13.656  0.597  14.447
sc_DTU_analysis               9.006  1.709   8.667
create_sce_from_dir           6.115  2.867   6.861
plot_isoform_heatmap          7.025  0.122   7.147
sc_long_pipeline              4.907  1.732   4.454
SingleCellPipeline            4.854  1.253   4.176
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

Status: 4 NOTEs
See
  ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.


Installation output

FLAMES.Rcheck/00install.out

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.3.4’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:122:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  122 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:163:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  163 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:187:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  187 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:295:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  295 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:354:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  354 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:356:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  356 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:357:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  357 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:396:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  396 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, std::ostream&)’:
main-functions/flexiplex.cpp:421:38: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  421 |                << (barcode.flank_end == std::string::npos ? "True" : "False")
      |                    ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, gzFile, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:462:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  462 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:477:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  477 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:482:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  482 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:740:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  740 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:745:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  745 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:747:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  747 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:749:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  749 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

Tests output

FLAMES.Rcheck/tests/testthat.Rout


R version 4.5.1 (2025-06-13) -- "Great Square Root"
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> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e076b009da1/config_file_69127.json 
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e0776ae6436/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077938a238/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077938a238/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e074011ac4d/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e074011ac4d/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e074011ac4d/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e074011ac4d/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e074d6a8ef6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e074d6a8ef6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077fa43327/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e077fa43327/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e077fa43327/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e077fa43327/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e074d6a8ef6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073248b0c0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e07204f41a9/config_file_69127.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Aug  6 22:42:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpb1YEyk/file10e07204f41a9/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.36] collected minimizers
[M::mm_idx_gen::0.002*1.25] sorted minimizers
[M::main::0.002*1.24] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.23] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.22] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.170*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e07204f41a9/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e07204f41a9/rps24.fa /tmp/Rtmpb1YEyk/file10e07204f41a9/fastq/sample1.fq.gz
[M::main] Real time: 0.171 sec; CPU: 0.171 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpb1YEyk/file10e07204f41a9/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.42] collected minimizers
[M::mm_idx_gen::0.002*1.28] sorted minimizers
[M::main::0.002*1.28] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.26] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.25] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e07204f41a9/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e07204f41a9/rps24.fa /tmp/Rtmpb1YEyk/file10e07204f41a9/fastq/sample2.fq.gz
[M::main] Real time: 0.171 sec; CPU: 0.172 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpb1YEyk/file10e07204f41a9/sample3_align2genome.bam
[M::mm_idx_gen::0.002*1.16] collected minimizers
[M::mm_idx_gen::0.002*1.12] sorted minimizers
[M::main::0.002*1.12] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.11] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.11] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.332*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e07204f41a9/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e07204f41a9/rps24.fa /tmp/Rtmpb1YEyk/file10e07204f41a9/fastq/sample3.fq.gz
[M::main] Real time: 0.333 sec; CPU: 0.333 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Aug  6 22:42:27 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[22:42:34] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:42:34] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:42:34] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:42:34] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:42:36] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[22:42:36] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.


  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:42:54 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpb1YEyk/file10e07204f41a9/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.22] collected minimizers
[M::mm_idx_gen::0.001*1.17] sorted minimizers
[M::main::0.001*1.17] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.16] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.16] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.082*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e07204f41a9/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e07204f41a9/fastq/sample1.fq.gz
[M::main] Real time: 0.082 sec; CPU: 0.083 sec; Peak RSS: 0.003 GB
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpb1YEyk/file10e07204f41a9/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.22] collected minimizers
[M::mm_idx_gen::0.001*1.18] sorted minimizers
[M::main::0.001*1.17] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.17] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.16] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.087*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e07204f41a9/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e07204f41a9/fastq/sample2.fq.gz
[M::main] Real time: 0.087 sec; CPU: 0.087 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpb1YEyk/file10e07204f41a9/sample3_realign2transcript.bam
[M::mm_idx_gen::0.002*1.38] collected minimizers
[M::mm_idx_gen::0.002*1.32] sorted minimizers
[M::main::0.002*1.32] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.31] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.31] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.164*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e07204f41a9/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e07204f41a9/fastq/sample3.fq.gz
[M::main] Real time: 0.164 sec; CPU: 0.164 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Aug  6 22:42:54 2025 ----------
2025-08-07T02:42:54.495999Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:42:54.496387Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07204f41a9/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:42:54.496399Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:42:54.496403Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:42:54.496465Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:42:54.496470Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:42:54.497937Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:42:54.498060Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-08-07T02:42:54.498081Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-08-07T02:42:54.498084Z  INFO oarfish::bulk: number of aligned reads : 96
2025-08-07T02:42:54.498096Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-07T02:42:54.498665Z  INFO oarfish: oarfish completed successfully.
2025-08-07T02:42:54.506060Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:42:54.506427Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07204f41a9/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:42:54.506438Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:42:54.506442Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:42:54.506529Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:42:54.506536Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:42:54.508016Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:42:54.508158Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-08-07T02:42:54.508185Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-08-07T02:42:54.508188Z  INFO oarfish::bulk: number of aligned reads : 95
2025-08-07T02:42:54.508191Z  INFO oarfish::bulk: number of unique alignments : 82
2025-08-07T02:42:54.508757Z  INFO oarfish: oarfish completed successfully.
2025-08-07T02:42:54.515649Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:42:54.516020Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07204f41a9/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:42:54.516028Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:42:54.516032Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:42:54.516096Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:42:54.516105Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:42:54.518806Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-08-07T02:42:54.518979Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-08-07T02:42:54.519007Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-08-07T02:42:54.519009Z  INFO oarfish::bulk: number of aligned reads : 179
2025-08-07T02:42:54.519012Z  INFO oarfish::bulk: number of unique alignments : 143
2025-08-07T02:42:54.519721Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e07187459f2/config_file_69127.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Aug  6 22:42:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpb1YEyk/file10e07187459f2/sample1_align2genome.bam
sample2 ->/tmp/Rtmpb1YEyk/file10e07187459f2/sample2_align2genome.bam
sample3 ->/tmp/Rtmpb1YEyk/file10e07187459f2/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Aug  6 22:43:17 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:43:35 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpb1YEyk/file10e07187459f2/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpb1YEyk/file10e07187459f2/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpb1YEyk/file10e07187459f2/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:43:57 2025 ----------
2025-08-07T02:43:57.241856Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:43:57.242248Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07187459f2/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:43:57.242261Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:43:57.242265Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:43:57.242324Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:43:57.242332Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:43:57.243906Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:43:57.244041Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-08-07T02:43:57.244072Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-08-07T02:43:57.244075Z  INFO oarfish::bulk: number of aligned reads : 96
2025-08-07T02:43:57.244078Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-07T02:43:57.244731Z  INFO oarfish: oarfish completed successfully.
2025-08-07T02:43:57.256696Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:43:57.257079Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07187459f2/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:43:57.257110Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:43:57.257114Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:43:57.257172Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:43:57.257177Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:43:57.258783Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:43:57.258927Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-08-07T02:43:57.258949Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-08-07T02:43:57.258952Z  INFO oarfish::bulk: number of aligned reads : 95
2025-08-07T02:43:57.258954Z  INFO oarfish::bulk: number of unique alignments : 82
2025-08-07T02:43:57.259609Z  INFO oarfish: oarfish completed successfully.
2025-08-07T02:43:57.271250Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:43:57.271629Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07187459f2/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:43:57.271638Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:43:57.271652Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:43:57.271713Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:43:57.271719Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:43:57.274438Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-08-07T02:43:57.274644Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-08-07T02:43:57.274676Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-08-07T02:43:57.274678Z  INFO oarfish::bulk: number of aligned reads : 179
2025-08-07T02:43:57.274681Z  INFO oarfish::bulk: number of unique alignments : 143
2025-08-07T02:43:57.275429Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e073c666781/config_file_69127.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Aug  6 22:43:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpb1YEyk/file10e073c666781/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.45] collected minimizers
[M::mm_idx_gen::0.002*1.31] sorted minimizers
[M::main::0.002*1.30] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.29] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.27] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073c666781/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073c666781/rps24.fa /tmp/Rtmpb1YEyk/file10e073c666781/fastq/sample1.fq.gz
[M::main] Real time: 0.171 sec; CPU: 0.172 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpb1YEyk/file10e073c666781/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.07] collected minimizers
[M::mm_idx_gen::0.002*1.05] sorted minimizers
[M::main::0.002*1.05] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.05] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.04] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073c666781/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073c666781/rps24.fa /tmp/Rtmpb1YEyk/file10e073c666781/fastq/sample2.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.172 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpb1YEyk/file10e073c666781/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.29] collected minimizers
[M::mm_idx_gen::0.002*1.20] sorted minimizers
[M::main::0.002*1.20] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.19] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.18] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.332*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073c666781/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073c666781/rps24.fa /tmp/Rtmpb1YEyk/file10e073c666781/fastq/sample3.fq.gz
[M::main] Real time: 0.333 sec; CPU: 0.333 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Aug  6 22:43:58 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:44:16 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpb1YEyk/file10e073c666781/sample1_realign2transcript.bam
[M::mm_idx_gen::0.002*1.35] collected minimizers
[M::mm_idx_gen::0.002*1.31] sorted minimizers
[M::main::0.002*1.31] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.30] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.29] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.070*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e073c666781/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073c666781/fastq/sample1.fq.gz
[M::main] Real time: 0.070 sec; CPU: 0.071 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpb1YEyk/file10e073c666781/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.22] collected minimizers
[M::mm_idx_gen::0.001*1.18] sorted minimizers
[M::main::0.001*1.18] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.17] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.17] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.070*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e073c666781/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073c666781/fastq/sample2.fq.gz
[M::main] Real time: 0.070 sec; CPU: 0.070 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpb1YEyk/file10e073c666781/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.04] collected minimizers
[M::mm_idx_gen::0.001*1.03] sorted minimizers
[M::main::0.001*1.03] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.03] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.03] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.136*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e073c666781/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073c666781/fastq/sample3.fq.gz
[M::main] Real time: 0.136 sec; CPU: 0.136 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Aug  6 22:44:17 2025 ----------
22:44:17 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e07b993693/config_file_69127.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Aug  6 22:44:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpb1YEyk/file10e07b993693/sample1_align2genome.bam
sample2 ->/tmp/Rtmpb1YEyk/file10e07b993693/sample2_align2genome.bam
sample3 ->/tmp/Rtmpb1YEyk/file10e07b993693/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Aug  6 22:44:41 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:44:59 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpb1YEyk/file10e07b993693/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpb1YEyk/file10e07b993693/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpb1YEyk/file10e07b993693/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:45:22 2025 ----------
22:45:22 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmpb1YEyk/file10e073c666781/sample1_realign2transcript.bam', '/tmp/Rtmpb1YEyk/file10e073c666781/sample2_realign2transcript.bam', '/tmp/Rtmpb1YEyk/file10e073c666781/sample3_realign2transcript.bam'] /tmp/Rtmpb1YEyk/file10e073c666781/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e0745dcee48/config_file_69127.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Aug  6 22:45:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpb1YEyk/file10e0745dcee48/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.73] collected minimizers
[M::mm_idx_gen::0.002*1.49] sorted minimizers
[M::main::0.002*1.49] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.46] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.44] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.172*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e0745dcee48/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e0745dcee48/rps24.fa /tmp/Rtmpb1YEyk/file10e0745dcee48/fastq/sample1.fq.gz
[M::main] Real time: 0.173 sec; CPU: 0.174 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpb1YEyk/file10e0745dcee48/sample2_align2genome.bam
[M::mm_idx_gen::0.002*1.20] collected minimizers
[M::mm_idx_gen::0.003*1.15] sorted minimizers
[M::main::0.003*1.15] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.14] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.13] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.172*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e0745dcee48/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e0745dcee48/rps24.fa /tmp/Rtmpb1YEyk/file10e0745dcee48/fastq/sample2.fq.gz
[M::main] Real time: 0.173 sec; CPU: 0.173 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpb1YEyk/file10e0745dcee48/sample3_align2genome.bam
[M::mm_idx_gen::0.003*1.37] collected minimizers
[M::mm_idx_gen::0.003*1.30] sorted minimizers
[M::main::0.003*1.29] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.28] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.27] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.333*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e0745dcee48/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e0745dcee48/rps24.fa /tmp/Rtmpb1YEyk/file10e0745dcee48/fastq/sample3.fq.gz
[M::main] Real time: 0.334 sec; CPU: 0.335 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Aug  6 22:45:24 2025 -------------
Inputs:  ['/tmp/Rtmpb1YEyk/file10e07b993693/sample1_realign2transcript.bam', '/tmp/Rtmpb1YEyk/file10e07b993693/sample2_realign2transcript.bam', '/tmp/Rtmpb1YEyk/file10e07b993693/sample3_realign2transcript.bam'] /tmp/Rtmpb1YEyk/file10e07b993693/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:45:25 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpb1YEyk/file10e0745dcee48/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.03] collected minimizers
[M::mm_idx_gen::0.002*1.03] sorted minimizers
[M::main::0.002*1.02] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.02] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.02] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.172*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e0745dcee48/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e0745dcee48/fastq/sample1.fq.gz
[M::main] Real time: 0.173 sec; CPU: 0.173 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpb1YEyk/file10e0745dcee48/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.30] collected minimizers
[M::mm_idx_gen::0.002*1.24] sorted minimizers
[M::main::0.002*1.23] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.22] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.21] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.172*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e0745dcee48/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e0745dcee48/fastq/sample2.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.173 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpb1YEyk/file10e0745dcee48/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.20] collected minimizers
[M::mm_idx_gen::0.002*1.15] sorted minimizers
[M::main::0.002*1.15] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.14] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.14] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.305*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e0745dcee48/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e0745dcee48/fastq/sample3.fq.gz
[M::main] Real time: 0.305 sec; CPU: 0.305 sec; Peak RSS: 0.006 GB
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Aug  6 22:45:25 2025 ----------
2025-08-07T02:45:25.899008Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:45:25.899531Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e0745dcee48/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-08-07T02:45:25.899545Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:45:25.899548Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:45:25.899627Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:45:25.899634Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-07T02:45:25.902188Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:45:25.902326Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-08-07T02:45:25.902351Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-08-07T02:45:25.902354Z  INFO oarfish::bulk: number of aligned reads : 98
2025-08-07T02:45:25.902356Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-07T02:45:25.902980Z  INFO oarfish: oarfish completed successfully.
2025-08-07T02:45:25.910921Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:45:25.911429Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e0745dcee48/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-08-07T02:45:25.911441Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:45:25.911445Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:45:25.911543Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:45:25.911550Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-07T02:45:25.914164Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:45:25.914299Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-08-07T02:45:25.914325Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-08-07T02:45:25.914328Z  INFO oarfish::bulk: number of aligned reads : 97
2025-08-07T02:45:25.914330Z  INFO oarfish::bulk: number of unique alignments : 79
2025-08-07T02:45:25.914950Z  INFO oarfish: oarfish completed successfully.
2025-08-07T02:45:25.922562Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:45:25.923042Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e0745dcee48/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-08-07T02:45:25.923051Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:45:25.923055Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:45:25.923137Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:45:25.923149Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-07T02:45:25.927649Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:45:25.927857Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-08-07T02:45:25.927891Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-08-07T02:45:25.927894Z  INFO oarfish::bulk: number of aligned reads : 187
2025-08-07T02:45:25.927897Z  INFO oarfish::bulk: number of unique alignments : 140
2025-08-07T02:45:25.928664Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e073163c4f7/config_file_69127.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Aug  6 22:45:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpb1YEyk/file10e073163c4f7/sample1_align2genome.bam
sample2 ->/tmp/Rtmpb1YEyk/file10e073163c4f7/sample2_align2genome.bam
sample3 ->/tmp/Rtmpb1YEyk/file10e073163c4f7/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Aug  6 22:45:49 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:45:49 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpb1YEyk/file10e073163c4f7/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpb1YEyk/file10e073163c4f7/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpb1YEyk/file10e073163c4f7/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:46:11 2025 ----------
2025-08-07T02:46:11.814669Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:46:11.815033Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e073163c4f7/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-08-07T02:46:11.815043Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:46:11.815046Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:46:11.815130Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:46:11.815139Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-07T02:46:11.817642Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:46:11.817784Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-08-07T02:46:11.817812Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-08-07T02:46:11.817815Z  INFO oarfish::bulk: number of aligned reads : 98
2025-08-07T02:46:11.817818Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-07T02:46:11.818420Z  INFO oarfish: oarfish completed successfully.
2025-08-07T02:46:11.827508Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:46:11.828015Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e073163c4f7/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-08-07T02:46:11.828025Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:46:11.828028Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:46:11.828114Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:46:11.828127Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-07T02:46:11.830788Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:46:11.830930Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-08-07T02:46:11.830956Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-08-07T02:46:11.830959Z  INFO oarfish::bulk: number of aligned reads : 97
2025-08-07T02:46:11.830961Z  INFO oarfish::bulk: number of unique alignments : 79
2025-08-07T02:46:11.831583Z  INFO oarfish: oarfish completed successfully.
2025-08-07T02:46:11.840979Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:46:11.841373Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e073163c4f7/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-08-07T02:46:11.841386Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:46:11.841389Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:46:11.841461Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:46:11.841468Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-07T02:46:11.845899Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-07T02:46:11.846079Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-08-07T02:46:11.846130Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-08-07T02:46:11.846133Z  INFO oarfish::bulk: number of aligned reads : 187
2025-08-07T02:46:11.846135Z  INFO oarfish::bulk: number of unique alignments : 140
2025-08-07T02:46:11.846846Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e0715c5565/config_file_69127.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Aug  6 22:46:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpb1YEyk/file10e0715c5565/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.58] collected minimizers
[M::mm_idx_gen::0.002*1.39] sorted minimizers
[M::main::0.002*1.39] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.37] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.35] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.169*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e0715c5565/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e0715c5565/rps24.fa /tmp/Rtmpb1YEyk/file10e0715c5565/fastq/sample1.fq.gz
[M::main] Real time: 0.169 sec; CPU: 0.170 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpb1YEyk/file10e0715c5565/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.35] collected minimizers
[M::mm_idx_gen::0.002*1.22] sorted minimizers
[M::main::0.002*1.22] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.20] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.19] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.172*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e0715c5565/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e0715c5565/rps24.fa /tmp/Rtmpb1YEyk/file10e0715c5565/fastq/sample2.fq.gz
[M::main] Real time: 0.173 sec; CPU: 0.173 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpb1YEyk/file10e0715c5565/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.76] collected minimizers
[M::mm_idx_gen::0.002*1.47] sorted minimizers
[M::main::0.002*1.47] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.44] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.41] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.330*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e0715c5565/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e0715c5565/rps24.fa /tmp/Rtmpb1YEyk/file10e0715c5565/fastq/sample3.fq.gz
[M::main] Real time: 0.330 sec; CPU: 0.331 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Aug  6 22:46:12 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:46:13 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpb1YEyk/file10e0715c5565/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.20] collected minimizers
[M::mm_idx_gen::0.002*1.15] sorted minimizers
[M::main::0.002*1.15] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.14] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.14] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.093*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e0715c5565/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e0715c5565/fastq/sample1.fq.gz
[M::main] Real time: 0.093 sec; CPU: 0.094 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpb1YEyk/file10e0715c5565/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.43] collected minimizers
[M::mm_idx_gen::0.002*1.34] sorted minimizers
[M::main::0.002*1.34] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.32] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.30] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.091*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e0715c5565/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e0715c5565/fastq/sample2.fq.gz
[M::main] Real time: 0.091 sec; CPU: 0.092 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpb1YEyk/file10e0715c5565/sample3_realign2transcript.bam
[M::mm_idx_gen::0.002*1.03] collected minimizers
[M::mm_idx_gen::0.002*1.02] sorted minimizers
[M::main::0.002*1.02] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*1.02] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*1.02] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.174*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e0715c5565/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e0715c5565/fastq/sample3.fq.gz
[M::main] Real time: 0.174 sec; CPU: 0.174 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Aug  6 22:46:14 2025 ----------
22:46:14 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e074b989994/config_file_69127.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Aug  6 22:46:15 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpb1YEyk/file10e074b989994/sample1_align2genome.bam
sample2 ->/tmp/Rtmpb1YEyk/file10e074b989994/sample2_align2genome.bam
sample3 ->/tmp/Rtmpb1YEyk/file10e074b989994/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Aug  6 22:46:38 2025 -------------
Inputs:  ['/tmp/Rtmpb1YEyk/file10e0715c5565/sample1_realign2transcript.bam', '/tmp/Rtmpb1YEyk/file10e0715c5565/sample2_realign2transcript.bam', '/tmp/Rtmpb1YEyk/file10e0715c5565/sample3_realign2transcript.bam'] /tmp/Rtmpb1YEyk/file10e0715c5565/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:46:39 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpb1YEyk/file10e074b989994/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpb1YEyk/file10e074b989994/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpb1YEyk/file10e074b989994/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:47:01 2025 ----------
22:47:01 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e0725537f7a/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:47:02 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e0725537f7a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:47:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb1YEyk/file10e0725537f7a/matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e0725537f7a/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.92] collected minimizers
[M::mm_idx_gen::0.002*0.71] sorted minimizers
[M::main::0.002*0.72] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.73] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.75] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.101*7.22] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/Rtmpb1YEyk/file10e0725537f7a/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e0725537f7a/rps24.fa /tmp/Rtmpb1YEyk/file10e0725537f7a/matched_reads.fastq.gz
[M::main] Real time: 0.102 sec; CPU: 0.732 sec; Peak RSS: 0.018 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Aug  6 22:47:02 2025 ----------------
22:47:02 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e0725537f7a/align2genome.bam'
Inputs:  ['/tmp/Rtmpb1YEyk/file10e074b989994/sample1_realign2transcript.bam', '/tmp/Rtmpb1YEyk/file10e074b989994/sample2_realign2transcript.bam', '/tmp/Rtmpb1YEyk/file10e074b989994/sample3_realign2transcript.bam'] /tmp/Rtmpb1YEyk/file10e074b989994/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.28gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.28gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39526.69Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:47:04 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:47:14 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e0725537f7a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e0725537f7a/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb1YEyk/file10e0725537f7a/matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e0725537f7a/realign2transcript.bam
[M::mm_idx_gen::0.001*1.88] collected minimizers
[M::mm_idx_gen::0.002*3.83] sorted minimizers
[M::main::0.002*3.79] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*3.69] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*3.59] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.049*6.33] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 8 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e0725537f7a/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e0725537f7a/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.049 sec; CPU: 0.310 sec; Peak RSS: 0.008 GB
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Wed Aug  6 22:47:14 2025 ----------
2025-08-07T02:47:14.889749Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:47:14.890183Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e0725537f7a/realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:47:14.890195Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:47:14.890199Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:47:14.890253Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:47:14.890259Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:47:14.897002Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e076f0b1783/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:47:16 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e076f0b1783/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:47:16 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e076f0b1783/matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e076f0b1783/align2genome.bam
-- Running step: gene_quantification @ Wed Aug  6 22:47:37 2025 ----------------
22:47:37 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e076f0b1783/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.84gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.84gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38760.64Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:47:38 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:47:48 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e076f0b1783/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e076f0b1783/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e076f0b1783/matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e076f0b1783/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:48:10 2025 ----------
2025-08-07T02:48:10.660432Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:48:10.660843Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e076f0b1783/realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:48:10.660855Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:48:10.660859Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:48:10.660923Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:48:10.660930Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:48:10.667722Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e073bbf0097/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:48:12 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073bbf0097/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:48:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb1YEyk/file10e073bbf0097/matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073bbf0097/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*1.08] collected minimizers
[M::mm_idx_gen::0.004*0.91] sorted minimizers
[M::main::0.004*0.91] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*0.92] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*0.92] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.110*7.12] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/Rtmpb1YEyk/file10e073bbf0097/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073bbf0097/rps24.fa /tmp/Rtmpb1YEyk/file10e073bbf0097/matched_reads.fastq.gz
[M::main] Real time: 0.111 sec; CPU: 0.787 sec; Peak RSS: 0.019 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Aug  6 22:48:12 2025 ----------------
22:48:13 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e073bbf0097/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.81gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.81gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38821.63Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:48:13 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:48:24 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e073bbf0097/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e073bbf0097/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb1YEyk/file10e073bbf0097/matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073bbf0097/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.30] collected minimizers
[M::mm_idx_gen::0.002*2.24] sorted minimizers
[M::main::0.002*2.23] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*2.19] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*2.16] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.043*6.10] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 8 --seed 2022 /tmp/Rtmpb1YEyk/file10e073bbf0097/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073bbf0097/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.044 sec; CPU: 0.264 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Wed Aug  6 22:48:24 2025 ----------
22:48:24 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e074ec5ba56/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:48:25 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e074ec5ba56/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:48:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e074ec5ba56/matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e074ec5ba56/align2genome.bam
-- Running step: gene_quantification @ Wed Aug  6 22:48:47 2025 ----------------
22:48:47 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e074ec5ba56/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.18gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39337.48Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:48:48 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:48:58 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e074ec5ba56/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e074ec5ba56/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e074ec5ba56/matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e074ec5ba56/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:49:20 2025 ----------
22:49:20 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e0725f1b49a/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:49:21 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e0725f1b49a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:49:21 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb1YEyk/file10e0725f1b49a/matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e0725f1b49a/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.98] collected minimizers
[M::mm_idx_gen::0.002*2.68] sorted minimizers
[M::main::0.002*2.66] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*2.54] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*2.45] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.095*7.19] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/Rtmpb1YEyk/file10e0725f1b49a/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e0725f1b49a/rps24.fa /tmp/Rtmpb1YEyk/file10e0725f1b49a/matched_reads.fastq.gz
[M::main] Real time: 0.096 sec; CPU: 0.685 sec; Peak RSS: 0.020 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Aug  6 22:49:21 2025 ----------------
22:49:21 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e0725f1b49a/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.24gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.24gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39245.02Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:49:22 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:49:23 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e0725f1b49a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e0725f1b49a/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb1YEyk/file10e0725f1b49a/matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e0725f1b49a/realign2transcript.bam
[M::mm_idx_gen::0.001*1.81] collected minimizers
[M::mm_idx_gen::0.002*3.32] sorted minimizers
[M::main::0.002*3.30] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*3.19] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*3.12] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.091*6.65] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 8 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e0725f1b49a/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e0725f1b49a/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.091 sec; CPU: 0.605 sec; Peak RSS: 0.009 GB
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Wed Aug  6 22:49:23 2025 ----------
2025-08-07T02:49:23.365451Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:49:23.365909Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e0725f1b49a/realign2transcript.bam, contains 10 reference sequences.
2025-08-07T02:49:23.365919Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:49:23.365923Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:49:23.366008Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:49:23.366016Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-07T02:49:23.375851Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e071864762b/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:49:25 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e071864762b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:49:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e071864762b/matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071864762b/align2genome.bam
-- Running step: gene_quantification @ Wed Aug  6 22:49:46 2025 ----------------
22:49:46 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e071864762b/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.28gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.28gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38714.84Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:49:47 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:49:47 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e071864762b/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e071864762b/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e071864762b/matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071864762b/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:50:09 2025 ----------
2025-08-07T02:50:09.426753Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:50:09.427206Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e071864762b/realign2transcript.bam, contains 10 reference sequences.
2025-08-07T02:50:09.427218Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:50:09.427222Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:50:09.427294Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:50:09.427303Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-07T02:50:09.437215Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e077e3b21da/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:50:11 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077e3b21da/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:50:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb1YEyk/file10e077e3b21da/matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e077e3b21da/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.32] collected minimizers
[M::mm_idx_gen::0.002*1.96] sorted minimizers
[M::main::0.002*1.95] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.90] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.85] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.086*7.09] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/Rtmpb1YEyk/file10e077e3b21da/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e077e3b21da/rps24.fa /tmp/Rtmpb1YEyk/file10e077e3b21da/matched_reads.fastq.gz
[M::main] Real time: 0.087 sec; CPU: 0.612 sec; Peak RSS: 0.019 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Aug  6 22:50:11 2025 ----------------
22:50:11 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e077e3b21da/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.15gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38593.30Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:50:12 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:50:13 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e077e3b21da/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e077e3b21da/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb1YEyk/file10e077e3b21da/matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e077e3b21da/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*2.05] collected minimizers
[M::mm_idx_gen::0.003*2.88] sorted minimizers
[M::main::0.003*2.86] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*2.79] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*2.74] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.050*6.25] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 8 --seed 2022 /tmp/Rtmpb1YEyk/file10e077e3b21da/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e077e3b21da/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.050 sec; CPU: 0.313 sec; Peak RSS: 0.008 GB
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Wed Aug  6 22:50:13 2025 ----------
22:50:13 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e0766b6e65b/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:50:14 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e0766b6e65b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:50:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e0766b6e65b/matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e0766b6e65b/align2genome.bam
-- Running step: gene_quantification @ Wed Aug  6 22:50:36 2025 ----------------
22:50:36 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e0766b6e65b/align2genome.bam'
	Counter({'counted_reads': 358})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.90gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.90gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38460.23Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:50:37 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:50:38 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e0766b6e65b/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e0766b6e65b/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e0766b6e65b/matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e0766b6e65b/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:50:59 2025 ----------
22:50:59 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e07a9f615c/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:51:01 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e07a9f615c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e07a9f615c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e07a9f615c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e07a9f615c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:51:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.38] collected minimizers
[M::mm_idx_gen::0.002*1.26] sorted minimizers
[M::main::0.002*1.25] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.24] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.22] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.600*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e07a9f615c/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e07a9f615c/rps24.fa /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.600 sec; CPU: 0.601 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*1.52] collected minimizers
[M::mm_idx_gen::0.002*1.36] sorted minimizers
[M::main::0.002*1.35] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.33] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.32] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.152*1.01] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e07a9f615c/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e07a9f615c/rps24.fa /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.153 sec; CPU: 0.153 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.91] collected minimizers
[M::mm_idx_gen::0.002*0.94] sorted minimizers
[M::main::0.002*0.94] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.94] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.94] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.155*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e07a9f615c/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e07a9f615c/rps24.fa /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.155 sec; CPU: 0.155 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.12] collected minimizers
[M::mm_idx_gen::0.002*1.08] sorted minimizers
[M::main::0.002*1.08] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.07] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.07] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.299*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e07a9f615c/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e07a9f615c/rps24.fa /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.300 sec; CPU: 0.300 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Aug  6 22:51:03 2025 ----------------
22:51:03 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_align2genome.bam', and
'/tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.86gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39348.41Read/s]
parsing /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 189147.32Read/s]
parsing /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.53gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153181.89Read/s]
parsing /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 76683.93Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:51:04 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:51:27 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq, /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample1.fq.gz, /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample2.fq.gz, /tmp/Rtmpb1YEyk/file10e07a9f615c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_realign2transcript.bam
[M::mm_idx_gen::0.001*1.33] collected minimizers
[M::mm_idx_gen::0.001*1.26] sorted minimizers
[M::main::0.001*1.26] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.25] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.24] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.273*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e07a9f615c/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.273 sec; CPU: 0.274 sec; Peak RSS: 0.005 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.46] collected minimizers
[M::mm_idx_gen::0.001*1.36] sorted minimizers
[M::main::0.001*1.36] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.34] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.33] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.073*1.01] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e07a9f615c/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.073 sec; CPU: 0.073 sec; Peak RSS: 0.003 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.49] collected minimizers
[M::mm_idx_gen::0.001*1.39] sorted minimizers
[M::main::0.001*1.38] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.36] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.35] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.078*1.01] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e07a9f615c/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.078 sec; CPU: 0.078 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*0.91] collected minimizers
[M::mm_idx_gen::0.002*0.93] sorted minimizers
[M::main::0.002*0.93] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*0.93] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*0.93] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.135*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e07a9f615c/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.135 sec; CPU: 0.135 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Aug  6 22:51:28 2025 ----------
2025-08-07T02:51:28.350613Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:51:28.350996Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07a9f615c/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:51:28.351006Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:51:28.351009Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:51:28.351073Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:51:28.351079Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:51:28.356646Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:51:29.671512Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:51:29.671892Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07a9f615c/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:51:29.671912Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:51:29.671916Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:51:29.671974Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:51:29.671980Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:51:31.012125Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:51:31.012524Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07a9f615c/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:51:31.012533Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:51:31.012536Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:51:31.012604Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:51:31.012619Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:51:32.281083Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:51:32.281550Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e07a9f615c/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:51:32.281559Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:51:32.281562Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:51:32.281625Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:51:32.281641Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e077f086d85/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:51:33 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077f086d85/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077f086d85/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077f086d85/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077f086d85/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:51:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_align2genome.bam
/tmp/Rtmpb1YEyk/file10e077f086d85/sample1_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f086d85/sample1_align2genome.bam
/tmp/Rtmpb1YEyk/file10e077f086d85/sample2_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f086d85/sample2_align2genome.bam
/tmp/Rtmpb1YEyk/file10e077f086d85/sample3_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f086d85/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Aug  6 22:51:57 2025 ----------------
22:51:57 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e077f086d85/sample1_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e077f086d85/sample2_align2genome.bam', and
'/tmp/Rtmpb1YEyk/file10e077f086d85/sample3_align2genome.bam'
parsing /tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.38gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39086.45Read/s]
parsing /tmp/Rtmpb1YEyk/file10e077f086d85/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.72gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 186908.61Read/s]
parsing /tmp/Rtmpb1YEyk/file10e077f086d85/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.09gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 152179.26Read/s]
parsing /tmp/Rtmpb1YEyk/file10e077f086d85/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.10gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77087.57Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:51:58 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:52:21 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e077f086d85/fastq, /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample1.fq.gz, /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample2.fq.gz, /tmp/Rtmpb1YEyk/file10e077f086d85/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f086d85/sample1_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f086d85/sample2_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f086d85/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f086d85/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f086d85/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f086d85/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e077f086d85/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f086d85/sample1_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e077f086d85/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f086d85/sample2_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e077f086d85/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f086d85/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:52:43 2025 ----------
2025-08-07T02:52:43.314622Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:52:43.315083Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e077f086d85/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:52:43.315102Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:52:43.315105Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:52:43.315168Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:52:43.315175Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-07T02:52:43.321296Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:52:44.815770Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:52:44.816172Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e077f086d85/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:52:44.816184Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:52:44.816188Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:52:44.816253Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:52:44.816260Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:52:46.177728Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:52:46.178250Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e077f086d85/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:52:46.178261Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:52:46.178265Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:52:46.178320Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:52:46.178326Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:52:47.490247Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:52:47.490654Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e077f086d85/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-07T02:52:47.490664Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:52:47.490667Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:52:47.490733Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:52:47.490739Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e076509d29d/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:52:49 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e076509d29d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e076509d29d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e076509d29d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e076509d29d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:52:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.90] collected minimizers
[M::mm_idx_gen::0.002*0.93] sorted minimizers
[M::main::0.002*0.93] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.94] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.94] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.599*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e076509d29d/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e076509d29d/rps24.fa /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.599 sec; CPU: 0.599 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.51] collected minimizers
[M::mm_idx_gen::0.002*1.34] sorted minimizers
[M::main::0.002*1.34] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.32] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.30] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.153*1.00] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e076509d29d/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e076509d29d/rps24.fa /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.154 sec; CPU: 0.155 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.89] collected minimizers
[M::mm_idx_gen::0.002*0.93] sorted minimizers
[M::main::0.002*0.93] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.94] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.94] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.156*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e076509d29d/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e076509d29d/rps24.fa /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.156 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.91] collected minimizers
[M::mm_idx_gen::0.002*0.94] sorted minimizers
[M::main::0.002*0.94] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.95] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.95] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.298*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e076509d29d/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e076509d29d/rps24.fa /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.299 sec; CPU: 0.299 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Aug  6 22:52:51 2025 ----------------
22:52:51 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e076509d29d/sample1_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e076509d29d/sample2_align2genome.bam', and
'/tmp/Rtmpb1YEyk/file10e076509d29d/sample3_align2genome.bam'
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.69gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39429.04Read/s]
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.56gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 186851.99Read/s]
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 152735.64Read/s]
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.46gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77674.60Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:52:52 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
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  |====================================================                  |  75%
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  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:53:15 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e076509d29d/fastq, /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample1.fq.gz, /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample2.fq.gz, /tmp/Rtmpb1YEyk/file10e076509d29d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*0.89] collected minimizers
[M::mm_idx_gen::0.002*0.90] sorted minimizers
[M::main::0.002*0.90] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*0.90] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*0.91] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.230*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e076509d29d/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.231 sec; CPU: 0.230 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.18] collected minimizers
[M::mm_idx_gen::0.001*1.14] sorted minimizers
[M::main::0.001*1.14] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.13] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.13] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.059*1.00] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e076509d29d/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.060 sec; CPU: 0.060 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.63] collected minimizers
[M::mm_idx_gen::0.001*1.49] sorted minimizers
[M::main::0.001*1.49] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.47] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.45] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.064*1.01] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e076509d29d/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.064 sec; CPU: 0.064 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*0.97] collected minimizers
[M::mm_idx_gen::0.002*0.98] sorted minimizers
[M::main::0.002*0.98] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*0.98] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*0.98] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.117*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e076509d29d/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.117 sec; CPU: 0.117 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Aug  6 22:53:15 2025 ----------
22:53:15 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e076509d29d/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e076509d29d/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e076509d29d/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e076509d29d/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e076509d29d/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e075767b6ac/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:53:18 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e075767b6ac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e075767b6ac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e075767b6ac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e075767b6ac/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:53:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_align2genome.bam
/tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_align2genome.bam
/tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_align2genome.bam
/tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Aug  6 22:53:43 2025 ----------------
22:53:43 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_align2genome.bam', and
'/tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.61gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:704: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39473.28Read/s]
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.27gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 187644.46Read/s]
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.89gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 154159.28Read/s]
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.78gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77304.10Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:53:44 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:54:06 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq, /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample1.fq.gz, /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample2.fq.gz, /tmp/Rtmpb1YEyk/file10e075767b6ac/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:54:28 2025 ----------
22:54:28 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e075767b6ac/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e075767b6ac/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e075767b6ac/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e075767b6ac/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e073abfe949/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:54:32 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073abfe949/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073abfe949/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073abfe949/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073abfe949/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:54:33 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*1.27] collected minimizers
[M::mm_idx_gen::0.002*1.20] sorted minimizers
[M::main::0.002*1.20] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.18] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.18] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.598*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073abfe949/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073abfe949/rps24.fa /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.599 sec; CPU: 0.599 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.48] collected minimizers
[M::mm_idx_gen::0.002*1.32] sorted minimizers
[M::main::0.002*1.32] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.30] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.28] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.154*1.00] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073abfe949/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073abfe949/rps24.fa /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.154 sec; CPU: 0.155 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.21] collected minimizers
[M::mm_idx_gen::0.002*1.13] sorted minimizers
[M::main::0.002*1.13] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.12] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.12] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.156*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073abfe949/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073abfe949/rps24.fa /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.157 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*0.84] collected minimizers
[M::mm_idx_gen::0.002*0.90] sorted minimizers
[M::main::0.002*0.90] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.90] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.91] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.298*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073abfe949/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073abfe949/rps24.fa /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.299 sec; CPU: 0.299 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Aug  6 22:54:34 2025 ----------------
22:54:34 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e073abfe949/sample1_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e073abfe949/sample2_align2genome.bam', and
'/tmp/Rtmpb1YEyk/file10e073abfe949/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.10gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39410.37Read/s]
parsing /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.16gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 189753.17Read/s]
parsing /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.29gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153507.06Read/s]
parsing /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.25gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 76175.31Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:54:35 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:54:36 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e073abfe949/fastq, /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample1.fq.gz, /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample2.fq.gz, /tmp/Rtmpb1YEyk/file10e073abfe949/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_realign2transcript.bam
[M::mm_idx_gen::0.002*1.73] collected minimizers
[M::mm_idx_gen::0.002*1.56] sorted minimizers
[M::main::0.002*1.55] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.53] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.50] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.725*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e073abfe949/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.726 sec; CPU: 0.727 sec; Peak RSS: 0.007 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_realign2transcript.bam
[M::mm_idx_gen::0.002*0.80] collected minimizers
[M::mm_idx_gen::0.003*0.83] sorted minimizers
[M::main::0.003*0.83] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.003*0.84] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.003*0.84] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.197*1.00] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e073abfe949/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.198 sec; CPU: 0.197 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_realign2transcript.bam
[M::mm_idx_gen::0.003*1.35] collected minimizers
[M::mm_idx_gen::0.003*1.30] sorted minimizers
[M::main::0.003*1.30] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.003*1.29] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.003*1.28] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.202*1.00] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e073abfe949/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.202 sec; CPU: 0.203 sec; Peak RSS: 0.003 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*0.81] collected minimizers
[M::mm_idx_gen::0.002*0.86] sorted minimizers
[M::main::0.002*0.86] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*0.87] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*0.88] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.343*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmpb1YEyk/file10e073abfe949/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.343 sec; CPU: 0.343 sec; Peak RSS: 0.005 GB
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Aug  6 22:54:38 2025 ----------
2025-08-07T02:54:38.205852Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:54:38.206320Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e073abfe949/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-08-07T02:54:38.206333Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:54:38.206337Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:54:38.206422Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:54:38.206430Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-07T02:54:38.217961Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:54:39.675406Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:54:39.675908Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e073abfe949/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-08-07T02:54:39.675918Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:54:39.675921Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:54:39.676021Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:54:39.676029Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:54:41.170074Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:54:41.170524Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e073abfe949/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-08-07T02:54:41.170535Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:54:41.170539Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:54:41.170633Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:54:41.170641Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:54:42.606395Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:54:42.606841Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e073abfe949/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-08-07T02:54:42.606850Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:54:42.606853Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:54:42.606933Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:54:42.606941Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e077f2750a8/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:54:44 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077f2750a8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077f2750a8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077f2750a8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e077f2750a8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:54:45 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_align2genome.bam
/tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_align2genome.bam
/tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_align2genome.bam
/tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Aug  6 22:55:09 2025 ----------------
22:55:09 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_align2genome.bam', and
'/tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_align2genome.bam'
parsing /tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.21gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38849.10Read/s]
parsing /tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 184277.53Read/s]
parsing /tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.74gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 152404.87Read/s]
parsing /tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.31gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77129.53Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:55:09 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:55:10 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq, /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample1.fq.gz, /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample2.fq.gz, /tmp/Rtmpb1YEyk/file10e077f2750a8/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:55:35 2025 ----------
2025-08-07T02:55:35.915561Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:55:35.916187Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e077f2750a8/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-08-07T02:55:35.916201Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:55:35.916206Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:55:35.916292Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:55:35.916301Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-07T02:55:35.928608Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:55:37.741699Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:55:37.742147Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e077f2750a8/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-08-07T02:55:37.742160Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:55:37.742165Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:55:37.742265Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:55:37.742275Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:55:39.485491Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:55:39.485867Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e077f2750a8/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-08-07T02:55:39.485878Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:55:39.485882Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:55:39.485967Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:55:39.485988Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-07T02:55:41.130881Z  INFO oarfish: setting user-provided filter parameters.
2025-08-07T02:55:41.131268Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpb1YEyk/file10e077f2750a8/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-08-07T02:55:41.131280Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-07T02:55:41.131284Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-07T02:55:41.131373Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-07T02:55:41.131382Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e073c14ebd/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:55:42 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073c14ebd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073c14ebd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073c14ebd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e073c14ebd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:55:43 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.03] collected minimizers
[M::mm_idx_gen::0.002*1.02] sorted minimizers
[M::main::0.002*1.02] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.02] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.02] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.597*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073c14ebd/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073c14ebd/rps24.fa /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.598 sec; CPU: 0.598 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*1.28] collected minimizers
[M::mm_idx_gen::0.002*1.20] sorted minimizers
[M::main::0.002*1.20] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.19] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.18] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.152*1.00] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073c14ebd/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073c14ebd/rps24.fa /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.153 sec; CPU: 0.153 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.34] collected minimizers
[M::mm_idx_gen::0.002*1.23] sorted minimizers
[M::main::0.002*1.22] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.21] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.20] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.156*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073c14ebd/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073c14ebd/rps24.fa /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.157 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_matched_reads.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.86] collected minimizers
[M::mm_idx_gen::0.002*1.59] sorted minimizers
[M::main::0.002*1.59] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.55] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.52] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.298*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmpb1YEyk/file10e073c14ebd/reference.bed --junc-bonus 1 /tmp/Rtmpb1YEyk/file10e073c14ebd/rps24.fa /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.299 sec; CPU: 0.300 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Aug  6 22:55:45 2025 ----------------
22:55:45 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_align2genome.bam', and
'/tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_align2genome.bam'
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39020.27Read/s]
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.73gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 190055.83Read/s]
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.69gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153284.90Read/s]
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.10gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 76663.18Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:55:46 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:55:46 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq, /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample1.fq.gz, /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample2.fq.gz, /tmp/Rtmpb1YEyk/file10e073c14ebd/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.002*0.99] collected minimizers
[M::mm_idx_gen::0.003*0.99] sorted minimizers
[M::main::0.003*0.99] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.003*0.99] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.003*0.99] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.352*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e073c14ebd/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.353 sec; CPU: 0.352 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.13] collected minimizers
[M::mm_idx_gen::0.002*1.10] sorted minimizers
[M::main::0.002*1.10] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.10] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.09] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.092*1.00] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e073c14ebd/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.093 sec; CPU: 0.093 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.49] collected minimizers
[M::mm_idx_gen::0.002*1.39] sorted minimizers
[M::main::0.002*1.38] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.37] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.35] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.094*1.01] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e073c14ebd/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.094 sec; CPU: 0.095 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
[M::mm_idx_gen::0.001*1.65] collected minimizers
[M::mm_idx_gen::0.002*1.51] sorted minimizers
[M::main::0.002*1.51] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.48] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.46] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.177*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmpb1YEyk/file10e073c14ebd/transcript_assembly.fa /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.178 sec; CPU: 0.178 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Aug  6 22:55:47 2025 ----------
22:55:47 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e073c14ebd/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e073c14ebd/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e073c14ebd/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e073c14ebd/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpb1YEyk/file10e071cdb6213/config_file_69127.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Aug  6 22:55:50 2025 ----------------
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e071cdb6213/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e071cdb6213/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e071cdb6213/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpb1YEyk/file10e071cdb6213/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
-- Running step: genome_alignment @ Wed Aug  6 22:55:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_align2genome.bam
/tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_align2genome.bam
/tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_align2genome.bam
/tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_matched_reads.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Aug  6 22:56:14 2025 ----------------
22:56:14 Wed Aug 06 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_align2genome.bam',
'/tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_align2genome.bam', and
'/tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_align2genome.bam'
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 175, 'not_enough_coverage': 1})
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.44gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39450.85Read/s]
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.59gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 193033.27Read/s]
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.04gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 155926.72Read/s]
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.23gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77985.97Read/s]
-- Running step: isoform_identification @ Wed Aug  6 22:56:15 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Wed Aug  6 22:56:16 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq, /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample1.fq.gz, /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample2.fq.gz, /tmp/Rtmpb1YEyk/file10e071cdb6213/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_matched_reads.fastq.gz, /tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_realign2transcript.bam
/tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Aug  6 22:56:37 2025 ----------
22:56:37 Wed Aug 06 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_realign2transcript.bamdone
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e071cdb6213/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e071cdb6213/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e071cdb6213/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_realign2transcript.bam...
parsing /tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpb1YEyk/file10e071cdb6213/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 235 | SKIP 0 | PASS 37 ]

[ FAIL 0 | WARN 235 | SKIP 0 | PASS 37 ]
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 175, 'not_enough_coverage': 1})
> 
> proc.time()
   user  system elapsed 
819.565  58.665 876.924 

Example timings

FLAMES.Rcheck/FLAMES-Ex.timings

nameusersystemelapsed
BulkPipeline4.0320.2314.084
MultiSampleSCPipeline13.656 0.59714.447
SingleCellPipeline4.8541.2534.176
add_gene_counts0.3070.0110.319
annotation_to_fasta0.6730.0020.675
blaze 4.82618.27613.471
bulk_long_pipeline 2.78913.152 2.998
combine_sce2.1420.3262.467
config-set0.1520.0080.158
config0.1470.0060.153
controllers-set0.3410.0100.353
controllers0.2330.0070.241
convolution_filter0.0000.0000.001
create_config0.0040.0010.005
create_sce_from_dir6.1152.8676.861
create_se_from_dir2.8730.1142.987
cutadapt0.1140.0160.129
example_pipeline0.3740.0080.383
experiment2.5900.0672.654
filter_annotation0.4820.0020.484
filter_coverage1.4300.0331.465
find_barcode0.8300.1050.932
find_bin0.0060.0020.008
find_variants20.400 0.30219.929
get_coverage1.4130.0281.443
index_genome0.1550.0040.159
mutation_positions1.5820.0721.654
plot_coverage2.5930.0422.637
plot_demultiplex1.8120.1161.931
plot_demultiplex_raw1.0800.0331.113
plot_isoform_heatmap7.0250.1227.147
plot_isoform_reduced_dim22.626 0.30022.926
plot_isoforms2.9920.0042.996
resume_FLAMES2.8130.0782.889
run_FLAMES2.6070.0772.680
run_step1.0060.0361.043
sc_DTU_analysis9.0061.7098.667
sc_impute_transcript0.5850.0000.584
sc_long_multisample_pipeline13.118 5.92412.703
sc_long_pipeline4.9071.7324.454
sc_mutations3.1970.3292.833
show-FLAMESPipeline0.3200.0080.329
steps-set0.4330.0110.445
steps0.1350.0100.145
weight_transcripts0.0240.0030.027