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###
### Running command:
###
###   /home/biocbuild/bbs-3.23-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.23-bioc/R/site-library --timings FLAMES_2.6.0.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck’
* using R version 4.6.0 RC (2026-04-17 r89917)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0
* running under: Ubuntu 24.04.4 LTS
* using session charset: UTF-8
* current time: 2026-04-29 04:24:34 UTC
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.6.0’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 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-3.23-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
* checking C++ specification ... INFO
  specified C++17
* checking installed package size ... INFO
  installed size is  8.8Mb
  sub-directories of 1Mb or more:
    bin    4.6Mb
    data   1.9Mb
* 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
Namespace in Imports field not imported from: ‘scuttle’
  All declared Imports should be used.
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  ‘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’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
barcode_group: no visible global function definition for ‘new’
barcode_segment: no visible global function definition for ‘new’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
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_diversity: no visible global function definition for ‘as’
find_variants: no visible global function definition for ‘setNames’
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 ‘CB’
plot_demultiplex_raw: no visible binding for global variable ‘UB’
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 ‘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_isoforms: no visible binding for global variable ‘xmin’
plot_isoforms: no visible binding for global variable ‘xmax’
plot_isoforms: no visible binding for global variable ‘ymin’
plot_isoforms: no visible binding for global variable ‘ymax’
plot_isoforms: no visible binding for global variable ‘x’
plot_isoforms: no visible binding for global variable ‘xend’
plot_isoforms: no visible binding for global variable ‘y’
plot_isoforms: no visible binding for global variable ‘yend’
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 ‘setNames’
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_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
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.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
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_files’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
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’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
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:
  CB Count FSM_match FlankEditDist Freq Reads Sample Type UB UMI_count
  adj.p.value allele allele_count as bam_index barcode barcode_rank
  barcodes_file barcodes_files capture.output cell_total_reads
  chisq.test count counts_no_ins cumpct demultiplexed reads
  demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype 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 setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x xend xmax xmin y yend ymax ymin
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  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-3.23-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                         6.361 15.707  15.455
find_variants                20.885  0.555  20.425
plot_isoform_reduced_dim     20.122  0.216  20.345
bulk_long_pipeline            6.698 10.637   2.410
sc_long_multisample_pipeline  8.086  4.627   7.470
sc_plot_genotype             10.533  1.960  10.793
MultiSampleSCPipeline        10.279  0.733  11.627
sc_DTU_analysis               6.664  1.469   6.333
create_se_from_dir            5.182  0.161   5.331
plot_durations                5.068  0.094   5.151
create_sce_from_dir           3.484  1.593   3.403
resume_FLAMES                 4.938  0.097   5.026
* 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-3.23-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.23-bioc/R/bin/R CMD INSTALL FLAMES
###
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* installing to library ‘/home/biocbuild/bbs-3.23-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.6.0’
** using non-staged installation via StagedInstall field
** libs
specified C++17
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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:142:56: warning: comparison of integer expressions of different signedness: ‘__gnu_cxx::__alloc_traits<std::allocator<unsigned int>, unsigned int>::value_type’ {aka ‘unsigned int’} and ‘int’ [-Wsign-compare]
  142 |       if (j == (len2 - 1) && dist_holder[i * len2 + j] < best) {
main-functions/flexiplex.cpp: In function ‘void refine_matched_segments(const std::string&, const std::vector<Segment>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_set<std::__cxx11::basic_string<char> > >*, const std::vector<int>&, Barcode&, std::vector<int>&, std::vector<int>&)’:
main-functions/flexiplex.cpp:348:70: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘const int’ [-Wsign-compare]
  348 |         } else if (editDistance < best_edit_distance && editDistance <= s.max_edit_distance) {
      |                                                         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:356:30: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘const int’ [-Wsign-compare]
  356 |       if (best_edit_distance <= s.max_edit_distance && current_segment_unambiguous) {
      |           ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, bool, bool, bool, const std::vector<Segment>&, const std::unordered_map<std::__cxx11::basic_string<char>, BarcodeGroup>&)’:
main-functions/flexiplex.cpp:790:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const size_t’ {aka ‘const long unsigned int’} [-Wsign-compare]
  790 |   for (int b = 0; b < vec_size; b++) {
      |                   ~~^~~~~~~~~~
main-functions/flexiplex.cpp:803:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const size_t’ {aka ‘const long unsigned int’} [-Wsign-compare]
  803 |     for (int k = 0; k < vec_size; k++) {
      |                     ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-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-3.23-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-3.23-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.23-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-3.23-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-3.23-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Building oarfish with cargo
mkdir -p ../inst/bin
mkdir cargo_temp
(cargo install oarfish --root cargo_temp --bin oarfish --vers 0.8.0)
    Updating crates.io index
  Installing oarfish v0.8.0
    Updating crates.io index
     Locking 279 packages to latest compatible versions
      Adding generic-array v0.14.7 (available: v0.14.9)
      Adding indicatif v0.17.11 (available: v0.18.4)
      Adding needletail v0.6.3 (available: v0.7.3)
      Adding noodles-bam v0.78.0 (available: v0.88.0)
      Adding noodles-bgzf v0.38.0 (available: v0.46.0)
      Adding noodles-sam v0.74.0 (available: v0.84.0)
      Adding rand v0.9.4 (available: v0.10.1)
      Adding tabled v0.18.0 (available: v0.20.0)
      Adding typed-builder v0.21.2 (available: v0.23.2)
   Compiling libc v0.2.186
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   Compiling lexical-write-integer v1.0.6
   Compiling lexical-parse-integer v1.0.6
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   Compiling lexical-parse-float v1.0.6
   Compiling clap_builder v4.6.0
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   Compiling crossbeam-channel v0.5.15
   Compiling crossbeam-queue v0.3.12
   Compiling proc-macro-error-attr2 v2.0.0
   Compiling lexical-core v1.0.6
   Compiling block-buffer v0.10.4
   Compiling crypto-common v0.1.7
   Compiling crossbeam-deque v0.8.6
   Compiling digest v0.10.7
   Compiling getrandom v0.2.17
   Compiling console v0.15.11
   Compiling num_cpus v1.17.0
   Compiling crossbeam v0.8.4
   Compiling jobserver v0.1.34
   Compiling rand_core v0.6.4
   Compiling rand_core v0.9.5
   Compiling cc v1.2.61
   Compiling indicatif v0.17.11
   Compiling rayon v1.12.0
   Compiling tempfile v3.27.0
   Compiling flate2 v1.1.9
   Compiling regex-automata v0.4.14
   Compiling num-integer v0.1.46
   Compiling num-complex v0.4.6
   Compiling approx v0.5.1
   Compiling approx v0.3.2
   Compiling noisy_float v0.2.1
   Compiling chrono v0.4.44
   Compiling noodles-bgzf v0.38.0
   Compiling num-bigint v0.4.6
   Compiling num-iter v0.1.45
   Compiling alga v0.9.3
   Compiling zstd-sys v2.0.16+zstd.1.5.7
   Compiling liblzma-sys v0.3.13
   Compiling bzip2-sys v0.1.13+1.0.8
   Compiling libz-sys v1.1.28
   Compiling sha2-asm v0.6.4
   Compiling lz4-sys v1.11.1+lz4-1.10.0
   Compiling minimap2-sys v0.1.30+minimap2.2.30
   Compiling ndarray v0.16.1
   Compiling ndarray v0.17.2
   Compiling ndarray v0.15.6
   Compiling sha2 v0.10.9
   Compiling csv v1.4.0
   Compiling proc-macro-error2 v2.0.1
   Compiling num-rational v0.4.2
   Compiling liblzma v0.3.6
   Compiling bytemuck_derive v1.10.2
   Compiling serde_derive v1.0.228
   Compiling futures-macro v0.3.32
   Compiling async-trait v0.1.89
   Compiling async-stream-impl v0.3.6
   Compiling tracing-attributes v0.1.31
   Compiling thiserror-impl v1.0.69
   Compiling typed-builder-macro v0.21.2
   Compiling derive-new v0.6.0
   Compiling tabled_derive v0.10.0
   Compiling strum_macros v0.26.4
   Compiling clap_derive v4.6.1
   Compiling num v0.4.3
   Compiling async-stream v0.3.6
   Compiling ppv-lite86 v0.2.21
   Compiling hashbrown v0.14.5
   Compiling futures-util v0.3.32
   Compiling rand_chacha v0.3.1
   Compiling rand_chacha v0.9.0
   Compiling tabled v0.18.0
   Compiling rand v0.9.4
   Compiling rand v0.8.6
   Compiling tracing v0.1.44
   Compiling bytemuck v1.25.0
   Compiling typed-builder v0.21.2
   Compiling safe_arch v0.7.4
   Compiling wide v0.7.33
   Compiling bzip2 v0.4.4
   Compiling clap v4.6.1
   Compiling rand_distr v0.4.3
   Compiling bstr v1.12.1
   Compiling matchers v0.2.0
   Compiling regex v1.12.3
   Compiling tracing-subscriber v0.3.23
   Compiling noodles-core v0.17.0
   Compiling noodles-csi v0.46.0
   Compiling arrow-format v0.8.1
   Compiling bincode v1.3.3
   Compiling bio-types v1.0.4
   Compiling ndarray-stats v0.6.0
   Compiling noodles-sam v0.74.0
   Compiling simba v0.9.1
   Compiling sendable-swapvec v0.4.3
   Compiling kders v0.1.1
   Compiling futures-executor v0.3.32
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   Compiling parquet-format-safe v0.2.4
   Compiling sprs v0.11.4
   Compiling noodles-bam v0.78.0
   Compiling lz4 v1.28.1
   Compiling nalgebra v0.33.3
   Compiling zstd v0.12.4
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   Compiling needletail v0.6.3
   Compiling minimap2 v0.1.31+minimap2.2.30
   Compiling seqcol_rs v0.4.1
   Compiling statrs v0.18.0
   Compiling oarfish v0.8.0
    Finished `release` profile [optimized] target(s) in 49.13s
  Installing /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/cargo_temp/bin/oarfish
   Installed package `oarfish v0.8.0` (executable `oarfish`)
warning: be sure to add `/home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/cargo_temp/bin` to your PATH to be able to run the installed binaries
cp cargo_temp/bin/oarfish ../inst/bin/
cargo uninstall oarfish --root cargo_temp
    Removing cargo_temp/bin/oarfish
rm -rf cargo_temp
installing to /home/biocbuild/bbs-3.23-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)

R version 4.6.0 RC (2026-04-17 r89917) -- "Because it was There"
Copyright (C) 2026 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

R is free software and comes with ABSOLUTELY NO WARRANTY.
You are welcome to redistribute it under certain conditions.
Type 'license()' or 'licence()' for distribution details.

R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
Type 'q()' to quit R.

> # 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")
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a590ba403/config_file_3208842.json
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a590ba403/config_file_3208842.json
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a590ba403/config_file_3208842.json
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a5b949a11/config_file_3208842.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a57ee17ea/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a2207f6f4/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a2207f6f4/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a7e814bb5/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a7e814bb5/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a7e814bb5/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a7e814bb5/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a47294a09/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
2 transcripts found in the BAM file.
1(50%) transcripts failed the filter.
Failed transcripts account for 100 reads, out of 200(50%) reads in total.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a35e482e4/config_file_3208842.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 Apr 29 00:33:42 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpQE6taE/file30f68a35e482e4/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpQE6taE/file30f68a35e482e4/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpQE6taE/file30f68a35e482e4/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 29 00:33:44 2026 -------------
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 ---
-- Running step: read_realignment @ Wed Apr 29 00:34:09 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpQE6taE/file30f68a35e482e4/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpQE6taE/file30f68a35e482e4/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpQE6taE/file30f68a35e482e4/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Apr 29 00:34:10 2026 ----------
2026-04-29T04:34:10.240459Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:34:10.240866Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a35e482e4/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-29T04:34:10.240892Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:34:10.240900Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:34:10.240986Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:34:10.240998Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-29T04:34:10.245075Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-04-29T04:34:10.245289Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 686   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 4     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:34:10.245356Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-04-29T04:34:10.245369Z  INFO oarfish::bulk: number of aligned reads : 283
2026-04-29T04:34:10.245376Z  INFO oarfish::bulk: number of unique alignments : 235
2026-04-29T04:34:10.246227Z  INFO oarfish: oarfish completed successfully.
2026-04-29T04:34:10.254439Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:34:10.254838Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a35e482e4/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-29T04:34:10.254894Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:34:10.254902Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:34:10.254967Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:34:10.254987Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-29T04:34:10.258993Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-04-29T04:34:10.259246Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 695   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:34:10.259314Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-04-29T04:34:10.259323Z  INFO oarfish::bulk: number of aligned reads : 285
2026-04-29T04:34:10.259330Z  INFO oarfish::bulk: number of unique alignments : 235
2026-04-29T04:34:10.260242Z  INFO oarfish: oarfish completed successfully.
2026-04-29T04:34:10.268177Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:34:10.268592Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a35e482e4/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-29T04:34:10.268659Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:34:10.268668Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:34:10.268738Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:34:10.268751Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-29T04:34:10.272688Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-29T04:34:10.272909Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 696   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 284   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:34:10.272977Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-04-29T04:34:10.272985Z  INFO oarfish::bulk: number of aligned reads : 284
2026-04-29T04:34:10.272992Z  INFO oarfish::bulk: number of unique alignments : 237
2026-04-29T04:34:10.273890Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a7f4034a1/config_file_3208842.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 Apr 29 00:34:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpQE6taE/file30f68a7f4034a1/sample1_align2genome.bam
sample2 ->/tmp/RtmpQE6taE/file30f68a7f4034a1/sample2_align2genome.bam
sample3 ->/tmp/RtmpQE6taE/file30f68a7f4034a1/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Apr 29 00:34:32 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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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 ---
-- Running step: read_realignment @ Wed Apr 29 00:34:57 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpQE6taE/file30f68a7f4034a1/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpQE6taE/file30f68a7f4034a1/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpQE6taE/file30f68a7f4034a1/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:35:18 2026 ----------
2026-04-29T04:35:18.927354Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:35:18.927863Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a7f4034a1/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-29T04:35:18.927887Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:35:18.927924Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:35:18.927988Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:35:18.928000Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-29T04:35:18.932242Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-29T04:35:18.932477Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 702   │
│ aligned fraction too low        │ 13    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:35:18.932527Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-04-29T04:35:18.932535Z  INFO oarfish::bulk: number of aligned reads : 285
2026-04-29T04:35:18.932541Z  INFO oarfish::bulk: number of unique alignments : 238
2026-04-29T04:35:18.933418Z  INFO oarfish: oarfish completed successfully.
2026-04-29T04:35:18.942694Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:35:18.943052Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a7f4034a1/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-29T04:35:18.943074Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:35:18.943082Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:35:18.943173Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:35:18.943184Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-29T04:35:18.947152Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-29T04:35:18.947361Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 687   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 6     │
│ reads with valid best alignment │ 282   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:35:18.947433Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-04-29T04:35:18.947446Z  INFO oarfish::bulk: number of aligned reads : 282
2026-04-29T04:35:18.947453Z  INFO oarfish::bulk: number of unique alignments : 240
2026-04-29T04:35:18.948457Z  INFO oarfish: oarfish completed successfully.
2026-04-29T04:35:18.957861Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:35:18.958228Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a7f4034a1/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-29T04:35:18.958283Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:35:18.958292Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:35:18.958362Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:35:18.958381Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-29T04:35:18.962231Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-29T04:35:18.962430Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 688   │
│ aligned fraction too low        │ 15    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:35:18.962498Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-04-29T04:35:18.962506Z  INFO oarfish::bulk: number of aligned reads : 283
2026-04-29T04:35:18.962513Z  INFO oarfish::bulk: number of unique alignments : 240
2026-04-29T04:35:18.963650Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a4557bcd5/config_file_3208842.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 Apr 29 00:35:19 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpQE6taE/file30f68a4557bcd5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpQE6taE/file30f68a4557bcd5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpQE6taE/file30f68a4557bcd5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 29 00:35:21 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 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 ---
-- Running step: read_realignment @ Wed Apr 29 00:35:41 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpQE6taE/file30f68a4557bcd5/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpQE6taE/file30f68a4557bcd5/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpQE6taE/file30f68a4557bcd5/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 29 00:35:42 2026 ----------
00:35:42 Wed Apr 29 2026 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)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a7c44ac8d/config_file_3208842.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 Apr 29 00:35:44 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample1_align2genome.bam
sample2 ->/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample2_align2genome.bam
sample3 ->/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Apr 29 00:36:06 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  67%
  |                                                                            
  |======================================================================| 100%

Detected 5 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 ---
-- Running step: read_realignment @ Wed Apr 29 00:36:26 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:36:49 2026 ----------
00:36:49 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpQE6taE/file30f68a4557bcd5/sample1_realign2transcript.bam', '/tmp/RtmpQE6taE/file30f68a4557bcd5/sample2_realign2transcript.bam', '/tmp/RtmpQE6taE/file30f68a4557bcd5/sample3_realign2transcript.bam'] /tmp/RtmpQE6taE/file30f68a4557bcd5/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 866, 'not_enough_coverage': 30, 'unmapped': 4})
Inputs:  ['/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample1_realign2transcript.bam', '/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample2_realign2transcript.bam', '/tmp/RtmpQE6taE/file30f68a7c44ac8d/sample3_realign2transcript.bam'] /tmp/RtmpQE6taE/file30f68a7c44ac8d/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a22e4659a/config_file_3208842.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 Apr 29 00:36:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpQE6taE/file30f68a22e4659a/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpQE6taE/file30f68a22e4659a/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpQE6taE/file30f68a22e4659a/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 29 00:36:51 2026 -------------
	Counter({'counted_reads': 863, 'not_enough_coverage': 32, 'unmapped': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:36:52 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpQE6taE/file30f68a22e4659a/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpQE6taE/file30f68a22e4659a/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpQE6taE/file30f68a22e4659a/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Apr 29 00:36:55 2026 ----------
2026-04-29T04:36:55.252056Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:36:55.252470Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a22e4659a/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-04-29T04:36:55.252495Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:36:55.252539Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:36:55.252636Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:36:55.252653Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-29T04:36:55.263188Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-29T04:36:55.263437Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3436  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:36:55.263495Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-04-29T04:36:55.263503Z  INFO oarfish::bulk: number of aligned reads : 293
2026-04-29T04:36:55.263509Z  INFO oarfish::bulk: number of unique alignments : 198
2026-04-29T04:36:55.264440Z  INFO oarfish: oarfish completed successfully.
2026-04-29T04:36:55.272869Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:36:55.273284Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a22e4659a/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-04-29T04:36:55.273305Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:36:55.273313Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:36:55.273421Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:36:55.273438Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-29T04:36:55.283736Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-29T04:36:55.283967Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3402  │
│ aligned fraction too low        │ 8     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 292   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:36:55.284047Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-04-29T04:36:55.284055Z  INFO oarfish::bulk: number of aligned reads : 292
2026-04-29T04:36:55.284068Z  INFO oarfish::bulk: number of unique alignments : 190
2026-04-29T04:36:55.285122Z  INFO oarfish: oarfish completed successfully.
2026-04-29T04:36:55.292895Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:36:55.293284Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a22e4659a/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-04-29T04:36:55.293339Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:36:55.293346Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:36:55.293435Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:36:55.293457Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-04-29T04:36:55.303359Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-29T04:36:55.303586Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3342  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:36:55.303664Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-04-29T04:36:55.303672Z  INFO oarfish::bulk: number of aligned reads : 290
2026-04-29T04:36:55.303694Z  INFO oarfish::bulk: number of unique alignments : 192
2026-04-29T04:36:55.304594Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a16bed199/config_file_3208842.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 Apr 29 00:36:55 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpQE6taE/file30f68a16bed199/sample1_align2genome.bam
sample2 ->/tmp/RtmpQE6taE/file30f68a16bed199/sample2_align2genome.bam
sample3 ->/tmp/RtmpQE6taE/file30f68a16bed199/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Apr 29 00:37:16 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:37:16 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpQE6taE/file30f68a16bed199/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpQE6taE/file30f68a16bed199/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpQE6taE/file30f68a16bed199/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:37:39 2026 ----------
2026-04-29T04:37:39.157980Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:37:39.158431Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a16bed199/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-04-29T04:37:39.158454Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:37:39.158463Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:37:39.158566Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:37:39.158581Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-29T04:37:39.167654Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-29T04:37:39.167868Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3045  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:37:39.167948Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-04-29T04:37:39.167962Z  INFO oarfish::bulk: number of aligned reads : 293
2026-04-29T04:37:39.167969Z  INFO oarfish::bulk: number of unique alignments : 208
2026-04-29T04:37:39.169027Z  INFO oarfish: oarfish completed successfully.
2026-04-29T04:37:39.176396Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:37:39.176773Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a16bed199/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-04-29T04:37:39.176829Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:37:39.176838Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:37:39.176923Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:37:39.176948Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-29T04:37:39.186267Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-29T04:37:39.186490Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2896  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:37:39.186565Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-04-29T04:37:39.186573Z  INFO oarfish::bulk: number of aligned reads : 290
2026-04-29T04:37:39.186580Z  INFO oarfish::bulk: number of unique alignments : 198
2026-04-29T04:37:39.187556Z  INFO oarfish: oarfish completed successfully.
2026-04-29T04:37:39.194953Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:37:39.195323Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a16bed199/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-04-29T04:37:39.195380Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:37:39.195389Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:37:39.195477Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:37:39.195492Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-04-29T04:37:39.204788Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-29T04:37:39.204997Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2880  │
│ aligned fraction too low        │ 9     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 291   │
╰─────────────────────────────────┴───────╯

2026-04-29T04:37:39.205072Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-04-29T04:37:39.205080Z  INFO oarfish::bulk: number of aligned reads : 291
2026-04-29T04:37:39.205086Z  INFO oarfish::bulk: number of unique alignments : 206
2026-04-29T04:37:39.206045Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a13e780fb/config_file_3208842.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 Apr 29 00:37:39 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpQE6taE/file30f68a13e780fb/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpQE6taE/file30f68a13e780fb/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpQE6taE/file30f68a13e780fb/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Apr 29 00:37:41 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:37:41 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpQE6taE/file30f68a13e780fb/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpQE6taE/file30f68a13e780fb/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpQE6taE/file30f68a13e780fb/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 29 00:37:43 2026 ----------
00:37:43 Wed Apr 29 2026 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)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a6c218ba/config_file_3208842.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 Apr 29 00:37:44 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpQE6taE/file30f68a6c218ba/sample1_align2genome.bam
sample2 ->/tmp/RtmpQE6taE/file30f68a6c218ba/sample2_align2genome.bam
sample3 ->/tmp/RtmpQE6taE/file30f68a6c218ba/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Apr 29 00:38:04 2026 -------------
Inputs:  ['/tmp/RtmpQE6taE/file30f68a13e780fb/sample1_realign2transcript.bam', '/tmp/RtmpQE6taE/file30f68a13e780fb/sample2_realign2transcript.bam', '/tmp/RtmpQE6taE/file30f68a13e780fb/sample3_realign2transcript.bam'] /tmp/RtmpQE6taE/file30f68a13e780fb/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:38:05 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpQE6taE/file30f68a6c218ba/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpQE6taE/file30f68a6c218ba/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpQE6taE/file30f68a6c218ba/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:38:24 2026 ----------
00:38:24 Wed Apr 29 2026 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)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a48669bdb/config_file_3208842.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 Apr 29 00:38:25 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a48669bdb/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:38:25 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpQE6taE/file30f68a48669bdb/matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a48669bdb/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 29 00:38:26 2026 ----------------
00:38:26 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a48669bdb/align2genome.bam'
Inputs:  ['/tmp/RtmpQE6taE/file30f68a6c218ba/sample1_realign2transcript.bam', '/tmp/RtmpQE6taE/file30f68a6c218ba/sample2_realign2transcript.bam', '/tmp/RtmpQE6taE/file30f68a6c218ba/sample3_realign2transcript.bam'] /tmp/RtmpQE6taE/file30f68a6c218ba/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.50gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.49gene_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, 417909.21Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:38:27 2026 -------------
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 ---
-- Running step: read_realignment @ Wed Apr 29 00:38:37 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a48669bdb/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a48669bdb/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpQE6taE/file30f68a48669bdb/matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a48669bdb/realign2transcript.bam
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 Apr 29 00:38:37 2026 ----------
2026-04-29T04:38:37.479026Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:38:37.479544Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a48669bdb/realign2transcript.bam, contains 5 reference sequences.
2026-04-29T04:38:37.479607Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:38:37.479617Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:38:37.479691Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:38:37.479721Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-29T04:38:37.484400Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a7147a7c1/config_file_3208842.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 Apr 29 00:38:38 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a7147a7c1/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:38:38 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a7147a7c1/matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a7147a7c1/align2genome.bam
-- Running step: gene_quantification @ Wed Apr 29 00:38:58 2026 ----------------
00:38:58 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a7147a7c1/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.43gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.42gene_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, 364202.70Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:38:59 2026 -------------
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 ---
-- Running step: read_realignment @ Wed Apr 29 00:39:09 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a7147a7c1/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a7147a7c1/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a7147a7c1/matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a7147a7c1/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:39:29 2026 ----------
2026-04-29T04:39:29.058584Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:39:29.059123Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a7147a7c1/realign2transcript.bam, contains 5 reference sequences.
2026-04-29T04:39:29.059202Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:39:29.059212Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:39:29.059272Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:39:29.059296Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-29T04:39:29.064127Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a4a9c8a4c/config_file_3208842.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 Apr 29 00:39:29 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a4a9c8a4c/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:39:29 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpQE6taE/file30f68a4a9c8a4c/matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4a9c8a4c/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.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 29 00:39:30 2026 ----------------
00:39:30 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a4a9c8a4c/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.62gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.62gene_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, 392122.96Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:39:30 2026 -------------
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 ---
-- Running step: read_realignment @ Wed Apr 29 00:39:40 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a4a9c8a4c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a4a9c8a4c/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpQE6taE/file30f68a4a9c8a4c/matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4a9c8a4c/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.
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 Apr 29 00:39:40 2026 ----------
00:39:40 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a271a872e/config_file_3208842.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 Apr 29 00:39:41 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a271a872e/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:39:41 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a271a872e/matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a271a872e/align2genome.bam
-- Running step: gene_quantification @ Wed Apr 29 00:40:06 2026 ----------------
00:40:06 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a271a872e/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,  2.50gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.50gene_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, 424455.96Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:40:06 2026 -------------
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 ---
-- Running step: read_realignment @ Wed Apr 29 00:40:16 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a271a872e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a271a872e/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a271a872e/matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a271a872e/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:40:36 2026 ----------
00:40:36 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a4ba34b99/config_file_3208842.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 Apr 29 00:40:37 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a4ba34b99/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:40:38 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpQE6taE/file30f68a4ba34b99/matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4ba34b99/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 29 00:40:38 2026 ----------------
00:40:38 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a4ba34b99/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.35gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.34gene_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, 318793.63Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:40:39 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:40:39 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a4ba34b99/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a4ba34b99/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpQE6taE/file30f68a4ba34b99/matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4ba34b99/realign2transcript.bam
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 Apr 29 00:40:39 2026 ----------
2026-04-29T04:40:39.682359Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:40:39.682798Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a4ba34b99/realign2transcript.bam, contains 10 reference sequences.
2026-04-29T04:40:39.682824Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:40:39.682874Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:40:39.682955Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:40:39.682969Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-29T04:40:39.690821Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a3b1934fc/config_file_3208842.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 Apr 29 00:40:40 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a3b1934fc/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:40:40 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a3b1934fc/matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3b1934fc/align2genome.bam
-- Running step: gene_quantification @ Wed Apr 29 00:41:00 2026 ----------------
00:41:00 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a3b1934fc/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.71gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.70gene_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, 389255.33Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:41:00 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:41:01 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a3b1934fc/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a3b1934fc/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a3b1934fc/matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3b1934fc/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:41:20 2026 ----------
2026-04-29T04:41:20.281825Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:41:20.282263Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a3b1934fc/realign2transcript.bam, contains 10 reference sequences.
2026-04-29T04:41:20.282285Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:41:20.282293Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:41:20.282358Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:41:20.282395Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-29T04:41:20.289971Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a63562d93/config_file_3208842.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 Apr 29 00:41:21 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a63562d93/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:41:21 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpQE6taE/file30f68a63562d93/matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a63562d93/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.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 29 00:41:21 2026 ----------------
00:41:21 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a63562d93/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.76gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.75gene_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, 386244.29Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:41:22 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:41:22 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a63562d93/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a63562d93/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpQE6taE/file30f68a63562d93/matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a63562d93/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.
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 Apr 29 00:41:22 2026 ----------
00:41:22 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a3553fc01/config_file_3208842.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 Apr 29 00:41:23 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a3553fc01/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.23-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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:41:24 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a3553fc01/matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3553fc01/align2genome.bam
-- Running step: gene_quantification @ Wed Apr 29 00:41:42 2026 ----------------
00:41:42 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a3553fc01/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,  2.66gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.66gene_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, 381897.51Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:41:43 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:41:43 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a3553fc01/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a3553fc01/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a3553fc01/matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3553fc01/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:42:02 2026 ----------
00:42:02 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a1ffd42/config_file_3208842.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 Apr 29 00:42:04 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1ffd42/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1ffd42/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a1ffd42/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a1ffd42/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 927
Number of chimera reads: 3
All done!
Reads	Barcodes
26	1
23	3
22	1
21	3
20	1
19	1
18	1
16	2
15	1
14	2
13	3
12	5
11	2
10	6
9	9
8	3
7	6
6	11
5	8
4	8
3	39
2	19
1	2
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1ffd42/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1ffd42/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	5
6	2
5	7
4	5
3	19
2	27
1	53
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1ffd42/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1ffd42/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 279
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
7	5
6	4
5	1
4	15
3	12
2	23
1	65
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1ffd42/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1ffd42/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:42:05 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a1ffd42/sample1_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1ffd42/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a1ffd42/sample2_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1ffd42/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a1ffd42/sample3_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1ffd42/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 29 00:42:09 2026 ----------------
00:42:09 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a1ffd42/sample1_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a1ffd42/sample2_align2genome.bam', and
'/tmp/RtmpQE6taE/file30f68a1ffd42/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.18gene_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, 182787.02Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1ffd42/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.12gene_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, 557693.86Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1ffd42/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.83gene_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, 575855.90Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1ffd42/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.06gene_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, 424903.15Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:42:10 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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Detected 7 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 29 00:42:34 2026 -------------------
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1ffd42/fastq, /tmp/RtmpQE6taE/file30f68a1ffd42/fastq/sample1.fq.gz, /tmp/RtmpQE6taE/file30f68a1ffd42/fastq/sample2.fq.gz, /tmp/RtmpQE6taE/file30f68a1ffd42/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ffd42/sample1_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ffd42/sample2_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ffd42/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ffd42/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ffd42/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ffd42/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpQE6taE/file30f68a1ffd42/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1ffd42/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpQE6taE/file30f68a1ffd42/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1ffd42/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpQE6taE/file30f68a1ffd42/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1ffd42/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Apr 29 00:42:36 2026 ----------
2026-04-29T04:42:36.282714Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:42:36.283080Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a1ffd42/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-04-29T04:42:36.283101Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:42:36.283109Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:42:36.283186Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:42:36.283198Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-29T04:42:36.292235Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:42:36.578328Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:42:36.578732Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a1ffd42/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-04-29T04:42:36.578756Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:42:36.578764Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:42:36.578843Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:42:36.578855Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-29T04:42:36.582336Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:42:36.901084Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:42:36.901414Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a1ffd42/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-04-29T04:42:36.901469Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:42:36.901477Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:42:36.901532Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:42:36.901554Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-29T04:42:36.904916Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:42:37.176904Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:42:37.177239Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a1ffd42/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-04-29T04:42:37.177290Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:42:37.177298Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:42:37.177353Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:42:37.177391Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-29T04:42:37.181446Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a2fee8411/config_file_3208842.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 Apr 29 00:42:37 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a2fee8411/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a2fee8411/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a2fee8411/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a2fee8411/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 930
Number of chimera reads: 3
All done!
Reads	Barcodes
28	1
25	1
23	3
22	1
21	1
19	2
18	1
17	1
16	2
15	2
14	2
13	4
12	2
11	5
10	4
9	7
8	7
7	2
6	11
5	12
4	7
3	37
2	20
1	2
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a2fee8411/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a2fee8411/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 284
Number of chimera reads: 1
All done!
Reads	Barcodes
10	1
9	1
8	3
7	2
6	4
5	7
4	6
3	15
2	21
1	60
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a2fee8411/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a2fee8411/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 1
All done!
Reads	Barcodes
8	2
7	3
6	1
5	6
4	9
3	19
2	29
1	56
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a2fee8411/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a2fee8411/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:42:38 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_align2genome.bam
/tmp/RtmpQE6taE/file30f68a2fee8411/sample1_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a2fee8411/sample1_align2genome.bam
/tmp/RtmpQE6taE/file30f68a2fee8411/sample2_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a2fee8411/sample2_align2genome.bam
/tmp/RtmpQE6taE/file30f68a2fee8411/sample3_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a2fee8411/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Apr 29 00:43:00 2026 ----------------
00:43:00 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a2fee8411/sample1_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a2fee8411/sample2_align2genome.bam', and
'/tmp/RtmpQE6taE/file30f68a2fee8411/sample3_align2genome.bam'
parsing /tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.08gene_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, 170096.36Read/s]
parsing /tmp/RtmpQE6taE/file30f68a2fee8411/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.60gene_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, 578556.61Read/s]
parsing /tmp/RtmpQE6taE/file30f68a2fee8411/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 30.77gene_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, 533409.30Read/s]
parsing /tmp/RtmpQE6taE/file30f68a2fee8411/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.01gene_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, 419078.37Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:43:01 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 8 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 29 00:43:26 2026 -------------------
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a2fee8411/fastq, /tmp/RtmpQE6taE/file30f68a2fee8411/fastq/sample1.fq.gz, /tmp/RtmpQE6taE/file30f68a2fee8411/fastq/sample2.fq.gz, /tmp/RtmpQE6taE/file30f68a2fee8411/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a2fee8411/sample1_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a2fee8411/sample2_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a2fee8411/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a2fee8411/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a2fee8411/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a2fee8411/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a2fee8411/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a2fee8411/sample1_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a2fee8411/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a2fee8411/sample2_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a2fee8411/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a2fee8411/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:43:46 2026 ----------
2026-04-29T04:43:46.736074Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:43:46.736433Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a2fee8411/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-04-29T04:43:46.736455Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:43:46.736504Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:43:46.736562Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:43:46.736575Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-29T04:43:46.746047Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:43:47.117622Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:43:47.118005Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a2fee8411/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-04-29T04:43:47.118030Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:43:47.118039Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:43:47.118146Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:43:47.118158Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-29T04:43:47.121831Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:43:47.444590Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:43:47.445019Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a2fee8411/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-04-29T04:43:47.445043Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:43:47.445051Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:43:47.445152Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:43:47.445165Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-29T04:43:47.448836Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:43:47.777154Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:43:47.777599Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a2fee8411/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-04-29T04:43:47.777622Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:43:47.777630Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:43:47.777701Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:43:47.777748Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-04-29T04:43:47.782136Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a4c5a4836/config_file_3208842.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 Apr 29 00:43:48 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a4c5a4836/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 929
Number of chimera reads: 2
All done!
Reads	Barcodes
27	1
26	1
24	1
22	2
21	1
20	3
19	1
18	1
17	2
16	2
15	2
14	1
12	1
11	7
10	5
9	5
8	9
7	5
6	13
5	11
4	5
3	33
2	22
1	3
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a4c5a4836/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 281
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	3
7	2
6	5
5	3
4	9
3	13
2	31
1	55
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a4c5a4836/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 0
All done!
Reads	Barcodes
9	1
8	3
7	1
6	5
5	5
4	6
3	18
2	30
1	50
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a4c5a4836/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a4c5a4836/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:43:49 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4c5a4836/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4c5a4836/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4c5a4836/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4c5a4836/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 29 00:43:53 2026 ----------------
00:43:53 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_align2genome.bam', and
'/tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_align2genome.bam'
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.35gene_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, 138405.78Read/s]
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.08gene_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, 499964.72Read/s]
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.07gene_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, 478234.06Read/s]
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.01gene_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, 378410.68Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:43:54 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
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Detected 8 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)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Apr 29 00:44:17 2026 -------------------
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq, /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq/sample1.fq.gz, /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq/sample2.fq.gz, /tmp/RtmpQE6taE/file30f68a4c5a4836/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4c5a4836/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4c5a4836/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4c5a4836/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a4c5a4836/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 29 00:44:19 2026 ----------
00:44:19 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a4c5a4836/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a4c5a4836/sample1_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a4c5a4836/sample2_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a4c5a4836/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a1ddd3470/config_file_3208842.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 Apr 29 00:44:21 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1ddd3470/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 924
Number of chimera reads: 2
All done!
Reads	Barcodes
26	1
25	1
22	1
21	3
20	2
19	2
18	1
17	4
16	1
15	1
14	1
13	1
12	1
11	7
10	5
9	3
8	11
7	3
6	5
5	19
4	7
3	35
2	19
1	3
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1ddd3470/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 279
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	3
6	5
5	8
4	7
3	11
2	23
1	66
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1ddd3470/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 277
Number of chimera reads: 0
All done!
Reads	Barcodes
8	1
7	3
6	8
5	5
4	4
3	19
2	22
1	60
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1ddd3470/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1ddd3470/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:44:23 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_align2genome.bam
/tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_align2genome.bam
/tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_align2genome.bam
/tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Apr 29 00:44:45 2026 ----------------
00:44:46 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_align2genome.bam', and
'/tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 5})
	Counter({'counted_reads': 262, 'not_enough_coverage': 8, 'unmapped': 2})
	Counter({'counted_reads': 264, 'not_enough_coverage': 8, 'unmapped': 1})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.47gene_group/s]
/home/biocbuild/bbs-3.23-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, 170369.96Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 30.19gene_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, 506656.36Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 30.09gene_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, 552056.44Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.53gene_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, 374050.58Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:44:47 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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  |===================================                                   |  50%
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  |====================================================                  |  75%
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  |======================================================================| 100%

Detected 8 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 ---
-- Running step: read_realignment @ Wed Apr 29 00:45:14 2026 -------------------
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq, /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq/sample1.fq.gz, /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq/sample2.fq.gz, /tmp/RtmpQE6taE/file30f68a1ddd3470/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:45:36 2026 ----------
00:45:36 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1ddd3470/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1ddd3470/sample1_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1ddd3470/sample2_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1ddd3470/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a47988931/config_file_3208842.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 Apr 29 00:45:38 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a47988931/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a47988931/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a47988931/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a47988931/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 928
Number of chimera reads: 2
All done!
Reads	Barcodes
28	1
26	1
21	2
20	3
19	4
17	2
14	4
13	2
12	5
11	7
10	1
9	6
8	4
7	7
6	12
5	15
4	4
3	35
2	19
1	3
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a47988931/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a47988931/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	4
6	3
5	5
4	8
3	12
2	36
1	54
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a47988931/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a47988931/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 282
Number of chimera reads: 0
All done!
Reads	Barcodes
9	2
7	1
6	4
5	8
4	14
3	9
2	27
1	59
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a47988931/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a47988931/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:45:39 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpQE6taE/file30f68a47988931/sampleA_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a47988931/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a47988931/sample1_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a47988931/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a47988931/sample2_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a47988931/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a47988931/sample3_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a47988931/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 29 00:45:43 2026 ----------------
00:45:43 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a47988931/sampleA_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a47988931/sample1_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a47988931/sample2_align2genome.bam', and
'/tmp/RtmpQE6taE/file30f68a47988931/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 5})
	Counter({'counted_reads': 265, 'not_enough_coverage': 8, 'unmapped': 2})
	Counter({'counted_reads': 261, 'not_enough_coverage': 9, 'unmapped': 1})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
parsing /tmp/RtmpQE6taE/file30f68a47988931/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.77gene_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, 149057.67Read/s]
parsing /tmp/RtmpQE6taE/file30f68a47988931/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.02gene_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, 468448.89Read/s]
parsing /tmp/RtmpQE6taE/file30f68a47988931/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.68gene_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, 433923.44Read/s]
parsing /tmp/RtmpQE6taE/file30f68a47988931/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.93gene_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, 371927.78Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:45:44 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:45:44 2026 -------------------
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a47988931/fastq, /tmp/RtmpQE6taE/file30f68a47988931/fastq/sample1.fq.gz, /tmp/RtmpQE6taE/file30f68a47988931/fastq/sample2.fq.gz, /tmp/RtmpQE6taE/file30f68a47988931/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a47988931/sampleA_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a47988931/sample1_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a47988931/sample2_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a47988931/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a47988931/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a47988931/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a47988931/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a47988931/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpQE6taE/file30f68a47988931/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a47988931/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpQE6taE/file30f68a47988931/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a47988931/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpQE6taE/file30f68a47988931/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a47988931/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpQE6taE/file30f68a47988931/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a47988931/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Apr 29 00:45:52 2026 ----------
2026-04-29T04:45:52.497662Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:45:52.498211Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a47988931/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-04-29T04:45:52.498237Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:45:52.498246Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:45:52.498601Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:45:52.498624Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-29T04:45:52.537309Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:45:53.277885Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:45:53.278431Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a47988931/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-04-29T04:45:53.278455Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:45:53.278464Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:45:53.278573Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:45:53.278592Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-29T04:45:53.292321Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:45:53.899886Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:45:53.900419Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a47988931/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-04-29T04:45:53.900445Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:45:53.900454Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:45:53.900568Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:45:53.900588Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-29T04:45:53.914274Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:45:54.544565Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:45:54.545197Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a47988931/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-04-29T04:45:54.545224Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:45:54.545235Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:45:54.545359Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:45:54.545379Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-04-29T04:45:54.561909Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a3c7ed4fa/config_file_3208842.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 Apr 29 00:45:55 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a3c7ed4fa/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 932
Number of chimera reads: 3
All done!
Reads	Barcodes
26	1
25	1
24	1
22	2
21	1
20	4
19	1
18	1
15	3
14	1
13	1
12	3
11	9
10	5
9	5
8	6
7	3
6	15
5	10
4	6
3	37
2	20
1	1
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a3c7ed4fa/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 1
All done!
Reads	Barcodes
8	2
7	4
6	5
5	2
4	9
3	18
2	27
1	56
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a3c7ed4fa/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 284
Number of chimera reads: 1
All done!
Reads	Barcodes
8	3
7	4
6	4
5	3
4	9
3	17
2	26
1	58
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a3c7ed4fa/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a3c7ed4fa/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:45:56 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_align2genome.bam
/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_align2genome.bam
/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_align2genome.bam
/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Apr 29 00:46:19 2026 ----------------
00:46:19 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_align2genome.bam', and
'/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_align2genome.bam'
parsing /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.34gene_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, 153899.08Read/s]
parsing /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.43gene_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, 501183.44Read/s]
parsing /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.02gene_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, 454164.93Read/s]
parsing /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.96gene_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, 368153.92Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:46:20 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:46:21 2026 -------------------
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq/sample1.fq.gz, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq/sample2.fq.gz, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:46:48 2026 ----------
2026-04-29T04:46:48.955731Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:46:48.956158Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-04-29T04:46:48.956185Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:46:48.956195Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:46:48.956311Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:46:48.956332Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-29T04:46:48.995444Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:46:49.735235Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:46:49.735863Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-04-29T04:46:49.735892Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:46:49.735902Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:46:49.736029Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:46:49.736050Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-29T04:46:49.749465Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:46:50.418257Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:46:50.418667Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-04-29T04:46:50.418704Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:46:50.418714Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:46:50.418832Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:46:50.418853Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-29T04:46:50.433194Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-29T04:46:51.185107Z  INFO oarfish: setting user-provided filter parameters.
2026-04-29T04:46:51.185684Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpQE6taE/file30f68a3c7ed4fa/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-04-29T04:46:51.185713Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-29T04:46:51.185722Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-29T04:46:51.185848Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-29T04:46:51.185870Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-04-29T04:46:51.202869Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a1406125/config_file_3208842.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 Apr 29 00:46:52 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1406125/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1406125/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a1406125/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a1406125/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 928
Number of chimera reads: 2
All done!
Reads	Barcodes
26	1
23	2
22	1
21	1
20	2
19	2
18	4
16	2
15	1
14	1
13	1
12	5
11	6
10	5
9	4
8	7
7	3
6	15
5	11
4	2
3	35
2	23
1	3
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1406125/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1406125/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 0
All done!
Reads	Barcodes
7	4
6	3
5	9
4	11
3	13
2	23
1	59
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1406125/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1406125/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 282
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	5
6	2
5	8
4	7
3	14
2	32
1	46
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1406125/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1406125/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:46:53 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpQE6taE/file30f68a1406125/sampleA_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1406125/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a1406125/sample1_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1406125/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a1406125/sample2_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1406125/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpQE6taE/file30f68a1406125/sample3_matched_reads.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1406125/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Apr 29 00:46:56 2026 ----------------
00:46:56 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a1406125/sampleA_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a1406125/sample1_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a1406125/sample2_align2genome.bam', and
'/tmp/RtmpQE6taE/file30f68a1406125/sample3_align2genome.bam'
parsing /tmp/RtmpQE6taE/file30f68a1406125/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.41gene_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, 169966.77Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.31gene_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, 512400.31Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.86gene_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, 538753.53Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.61gene_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, 369425.03Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:46:58 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:46:58 2026 -------------------
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1406125/fastq, /tmp/RtmpQE6taE/file30f68a1406125/fastq/sample1.fq.gz, /tmp/RtmpQE6taE/file30f68a1406125/fastq/sample2.fq.gz, /tmp/RtmpQE6taE/file30f68a1406125/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1406125/sampleA_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1406125/sample1_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1406125/sample2_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1406125/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1406125/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1406125/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1406125/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1406125/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpQE6taE/file30f68a1406125/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1406125/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpQE6taE/file30f68a1406125/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1406125/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpQE6taE/file30f68a1406125/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1406125/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/RtmpQE6taE/file30f68a1406125/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpQE6taE/file30f68a1406125/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.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Apr 29 00:47:01 2026 ----------
00:47:01 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpQE6taE/file30f68a1406125/sampleA_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1406125/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1406125/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample1_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1406125/sample1_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample2_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1406125/sample2_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample3_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1406125/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1406125/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpQE6taE/file30f68a1c27930c/config_file_3208842.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 Apr 29 00:47:05 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1c27930c/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1c27930c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a1c27930c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpQE6taE/file30f68a1c27930c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 929
Number of chimera reads: 2
All done!
Reads	Barcodes
29	1
24	1
22	1
21	2
20	3
19	1
18	2
16	4
15	1
14	1
13	5
11	4
10	7
9	5
8	9
7	3
6	13
5	14
4	2
3	32
2	20
1	6
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1c27930c/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1c27930c/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 281
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	3
7	1
6	4
5	5
4	8
3	16
2	33
1	50
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1c27930c/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1c27930c/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 0
All done!
Reads	Barcodes
10	1
9	1
7	1
6	4
5	10
4	7
3	16
2	28
1	51
Loading known barcodes from /tmp/RtmpQE6taE/file30f68a1c27930c/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpQE6taE/file30f68a1c27930c/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 chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Apr 29 00:47:06 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_align2genome.bam
/tmp/RtmpQE6taE/file30f68a1c27930c/sample1_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1c27930c/sample1_align2genome.bam
/tmp/RtmpQE6taE/file30f68a1c27930c/sample2_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1c27930c/sample2_align2genome.bam
/tmp/RtmpQE6taE/file30f68a1c27930c/sample3_matched_reads.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1c27930c/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Apr 29 00:47:29 2026 ----------------
00:47:29 Wed Apr 29 2026 quantify genes 
Using BAM(s): '/tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a1c27930c/sample1_align2genome.bam',
'/tmp/RtmpQE6taE/file30f68a1c27930c/sample2_align2genome.bam', and
'/tmp/RtmpQE6taE/file30f68a1c27930c/sample3_align2genome.bam'
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 275})
	Counter({'counted_reads': 275, 'not_enough_coverage': 1})
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.38gene_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, 138590.54Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.06gene_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, 490424.21Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.96gene_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, 504778.32Read/s]
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.00gene_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, 400372.66Read/s]
-- Running step: isoform_identification @ Wed Apr 29 00:47:30 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Apr 29 00:47:31 2026 -------------------
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1c27930c/fastq, /tmp/RtmpQE6taE/file30f68a1c27930c/fastq/sample1.fq.gz, /tmp/RtmpQE6taE/file30f68a1c27930c/fastq/sample2.fq.gz, /tmp/RtmpQE6taE/file30f68a1c27930c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1c27930c/sample1_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1c27930c/sample2_matched_reads.fastq.gz, /tmp/RtmpQE6taE/file30f68a1c27930c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1c27930c/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1c27930c/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpQE6taE/file30f68a1c27930c/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a1c27930c/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1c27930c/sample1_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a1c27930c/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1c27930c/sample2_realign2transcript.bam
/tmp/RtmpQE6taE/file30f68a1c27930c/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpQE6taE/file30f68a1c27930c/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Apr 29 00:47:53 2026 ----------
00:47:53 Wed Apr 29 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1c27930c/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample1_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1c27930c/sample1_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample2_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1c27930c/sample2_realign2transcript.bamdone
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample3_realign2transcript.bam...
parsing /tmp/RtmpQE6taE/file30f68a1c27930c/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpQE6taE/file30f68a1c27930c/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Filtering for genes with at least 2 detected isoforms ...
	6 isoform(s) left.

Aggregating counts by cluster labels ...

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

Filtering for genes with at least 2 isoforms expressing more than 1 counts ...
	3 gene(s), 6 transcript(s) left.

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

[ FAIL 0 | WARN 182 | SKIP 0 | PASS 98 ]

[ FAIL 0 | WARN 182 | SKIP 0 | PASS 98 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 275})
	Counter({'counted_reads': 272})
	Counter({'counted_reads': 358})
> 
> proc.time()
   user  system elapsed 
840.406  57.558 881.040