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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: 2026-05-08 04:01:59 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                         6.817 18.206  17.427
find_variants                21.458  1.312  21.904
plot_isoform_reduced_dim     21.767  0.748  22.527
bulk_long_pipeline            7.130 13.483   2.531
sc_long_multisample_pipeline  8.652  6.102   8.434
sc_plot_genotype             10.785  1.563  10.534
MultiSampleSCPipeline        10.943  0.780  12.011
sc_DTU_analysis               6.908  1.745   6.755
experiment                    6.842  0.201   8.156
create_sce_from_dir           3.783  2.412   3.884
create_se_from_dir            5.322  0.185   5.495
plot_durations                5.119  0.123   5.227
* 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: v0.89.0)
      Adding noodles-bgzf v0.38.0 (available: v0.47.0)
      Adding noodles-sam v0.74.0 (available: v0.85.0)
      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)
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   Compiling proc-macro-error-attr2 v2.0.0
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   Compiling lexical-core v1.0.6
   Compiling crossbeam-deque v0.8.6
   Compiling digest v0.10.7
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   Compiling jobserver v0.1.34
   Compiling getrandom v0.2.17
   Compiling console v0.15.11
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   Compiling rand_core v0.6.4
   Compiling cc v1.2.61
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   Compiling tempfile v3.27.0
   Compiling num-integer v0.1.46
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   Compiling approx v0.5.1
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   Compiling noisy_float v0.2.1
   Compiling chrono v0.4.44
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   Compiling num-iter v0.1.45
   Compiling csv v1.4.0
   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 minimap2-sys v0.1.30+minimap2.2.30
   Compiling lz4-sys v1.11.1+lz4-1.10.0
   Compiling alga v0.9.3
   Compiling ndarray v0.16.1
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   Compiling noodles-bgzf v0.38.0
   Compiling sha2 v0.10.9
   Compiling proc-macro-error2 v2.0.1
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   Compiling serde_derive v1.0.228
   Compiling futures-macro v0.3.32
   Compiling async-stream-impl v0.3.6
   Compiling tracing-attributes v0.1.31
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   Compiling thiserror-impl v1.0.69
   Compiling typed-builder-macro v0.21.2
   Compiling clap_derive v4.6.1
   Compiling strum_macros v0.26.4
   Compiling derive-new v0.6.0
   Compiling tabled_derive v0.10.0
   Compiling num-rational v0.4.2
   Compiling liblzma v0.3.6
   Compiling async-stream v0.3.6
   Compiling num v0.4.3
   Compiling futures-util v0.3.32
   Compiling tabled v0.18.0
   Compiling ppv-lite86 v0.2.21
   Compiling tracing v0.1.44
   Compiling bytemuck v1.25.0
   Compiling typed-builder v0.21.2
   Compiling safe_arch v0.7.4
   Compiling hashbrown v0.14.5
   Compiling rand_chacha v0.3.1
   Compiling rand_chacha v0.9.0
   Compiling clap v4.6.1
   Compiling bstr v1.12.1
   Compiling matchers v0.2.0
   Compiling regex v1.12.3
   Compiling rand v0.9.4
   Compiling tracing-subscriber v0.3.23
   Compiling rand v0.8.6
   Compiling wide v0.7.33
   Compiling noodles-core v0.17.0
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   Compiling rand_distr v0.4.3
   Compiling bzip2 v0.4.4
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   Compiling arrow-format v0.8.1
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   Compiling noodles-bam v0.78.0
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   Compiling minimap2 v0.1.31+minimap2.2.30
   Compiling statrs v0.18.0
   Compiling oarfish v0.8.0
    Finished `release` profile [optimized] target(s) in 49.69s
  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/RtmpzTR1Z4/file240961428d3ee4/config_file_2361697.json
i Writing configuration to: /tmp/RtmpzTR1Z4/file240961428d3ee4/config_file_2361697.json
i Writing configuration to: /tmp/RtmpzTR1Z4/file240961428d3ee4/config_file_2361697.json
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409613d8f46cf/config_file_2361697.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpzTR1Z4/file240961ad65f8b/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/RtmpzTR1Z4/file24096176652ee8/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/RtmpzTR1Z4/file24096176652ee8/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/RtmpzTR1Z4/file2409618d1b28d/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409618d1b28d/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409618d1b28d/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409618d1b28d/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/RtmpzTR1Z4/file240961415a2286/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/RtmpzTR1Z4/file240961666f1690/config_file_2361697.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 @ Fri May  8 00:11:29 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpzTR1Z4/file240961666f1690/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpzTR1Z4/file240961666f1690/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpzTR1Z4/file240961666f1690/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri May  8 00:11:31 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 @ Fri May  8 00:11:57 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpzTR1Z4/file240961666f1690/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpzTR1Z4/file240961666f1690/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpzTR1Z4/file240961666f1690/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri May  8 00:11:58 2026 ----------
2026-05-08T04:11:58.169924Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:11:58.170390Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file240961666f1690/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:11:58.170414Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:11:58.170422Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:11:58.170491Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:11:58.170503Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:11:58.174499Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-08T04:11:58.174716Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:11:58.174778Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-08T04:11:58.174791Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-08T04:11:58.174799Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-08T04:11:58.175664Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:11:58.182661Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:11:58.183094Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file240961666f1690/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:11:58.183138Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:11:58.183146Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:11:58.183201Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:11:58.183220Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:11:58.187207Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-08T04:11:58.187397Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:11:58.187459Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-05-08T04:11:58.187468Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-08T04:11:58.187475Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-08T04:11:58.188351Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:11:58.195484Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:11:58.195823Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file240961666f1690/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:11:58.195864Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:11:58.195872Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:11:58.195926Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:11:58.195937Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:11:58.199876Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-08T04:11:58.200060Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:11:58.200114Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-05-08T04:11:58.200122Z  INFO oarfish::bulk: number of aligned reads : 284
2026-05-08T04:11:58.200129Z  INFO oarfish::bulk: number of unique alignments : 237
2026-05-08T04:11:58.201008Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file24096143dea864/config_file_2361697.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 @ Fri May  8 00:11:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpzTR1Z4/file24096143dea864/sample1_align2genome.bam
sample2 ->/tmp/RtmpzTR1Z4/file24096143dea864/sample2_align2genome.bam
sample3 ->/tmp/RtmpzTR1Z4/file24096143dea864/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri May  8 00:12:20 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 @ Fri May  8 00:12:41 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpzTR1Z4/file24096143dea864/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpzTR1Z4/file24096143dea864/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpzTR1Z4/file24096143dea864/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:13:02 2026 ----------
2026-05-08T04:13:02.662052Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:13:02.662420Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file24096143dea864/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:13:02.662442Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:13:02.662473Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:13:02.662532Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:13:02.662543Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:13:02.666512Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-08T04:13:02.666710Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:13:02.666762Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-08T04:13:02.666770Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-08T04:13:02.666777Z  INFO oarfish::bulk: number of unique alignments : 238
2026-05-08T04:13:02.667604Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:13:02.679056Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:13:02.679444Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file24096143dea864/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:13:02.679465Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:13:02.679473Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:13:02.679549Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:13:02.679561Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:13:02.683491Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-08T04:13:02.683687Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:13:02.683755Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-05-08T04:13:02.683769Z  INFO oarfish::bulk: number of aligned reads : 282
2026-05-08T04:13:02.683777Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-08T04:13:02.684872Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:13:02.696278Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:13:02.696633Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file24096143dea864/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:13:02.696691Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:13:02.696700Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:13:02.696754Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:13:02.696772Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:13:02.700660Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-08T04:13:02.700860Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:13:02.700933Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-05-08T04:13:02.700941Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-08T04:13:02.700948Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-08T04:13:02.702210Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file240961502ea9ec/config_file_2361697.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 @ Fri May  8 00:13:02 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpzTR1Z4/file240961502ea9ec/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpzTR1Z4/file240961502ea9ec/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpzTR1Z4/file240961502ea9ec/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri May  8 00:13:04 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 @ Fri May  8 00:13:24 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpzTR1Z4/file240961502ea9ec/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpzTR1Z4/file240961502ea9ec/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpzTR1Z4/file240961502ea9ec/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri May  8 00:13:25 2026 ----------
00:13:25 Fri May 08 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/RtmpzTR1Z4/file240961748f1cc6/config_file_2361697.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 @ Fri May  8 00:13:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpzTR1Z4/file240961748f1cc6/sample1_align2genome.bam
sample2 ->/tmp/RtmpzTR1Z4/file240961748f1cc6/sample2_align2genome.bam
sample3 ->/tmp/RtmpzTR1Z4/file240961748f1cc6/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri May  8 00:13:49 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 @ Fri May  8 00:14:13 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpzTR1Z4/file240961748f1cc6/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpzTR1Z4/file240961748f1cc6/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpzTR1Z4/file240961748f1cc6/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:14:32 2026 ----------
00:14:32 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpzTR1Z4/file240961502ea9ec/sample1_realign2transcript.bam', '/tmp/RtmpzTR1Z4/file240961502ea9ec/sample2_realign2transcript.bam', '/tmp/RtmpzTR1Z4/file240961502ea9ec/sample3_realign2transcript.bam'] /tmp/RtmpzTR1Z4/file240961502ea9ec/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 866, 'not_enough_coverage': 30, 'unmapped': 4})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409616216fddf/config_file_2361697.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 @ Fri May  8 00:14:33 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpzTR1Z4/file2409616216fddf/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpzTR1Z4/file2409616216fddf/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpzTR1Z4/file2409616216fddf/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri May  8 00:14:35 2026 -------------
Inputs:  ['/tmp/RtmpzTR1Z4/file240961748f1cc6/sample1_realign2transcript.bam', '/tmp/RtmpzTR1Z4/file240961748f1cc6/sample2_realign2transcript.bam', '/tmp/RtmpzTR1Z4/file240961748f1cc6/sample3_realign2transcript.bam'] /tmp/RtmpzTR1Z4/file240961748f1cc6/transcript_assembly.fa.fai 5 0.4 0.4
	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 @ Fri May  8 00:14:35 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpzTR1Z4/file2409616216fddf/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpzTR1Z4/file2409616216fddf/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpzTR1Z4/file2409616216fddf/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri May  8 00:14:38 2026 ----------
2026-05-08T04:14:38.790859Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:14:38.791207Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409616216fddf/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-05-08T04:14:38.791228Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:14:38.791263Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:14:38.791352Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:14:38.791368Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-08T04:14:38.801322Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:14:38.801556Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:14:38.801614Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-05-08T04:14:38.801622Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-08T04:14:38.801629Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-08T04:14:38.802500Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:14:38.810255Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:14:38.810750Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409616216fddf/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-05-08T04:14:38.810773Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:14:38.810781Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:14:38.810887Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:14:38.810904Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-08T04:14:38.821436Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:14:38.821688Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:14:38.821761Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-05-08T04:14:38.821770Z  INFO oarfish::bulk: number of aligned reads : 292
2026-05-08T04:14:38.821782Z  INFO oarfish::bulk: number of unique alignments : 190
2026-05-08T04:14:38.822773Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:14:38.830445Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:14:38.830795Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409616216fddf/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-05-08T04:14:38.830839Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:14:38.830848Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:14:38.830929Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:14:38.830951Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-08T04:14:38.840917Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:14:38.841144Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:14:38.841215Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-05-08T04:14:38.841222Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-08T04:14:38.841229Z  INFO oarfish::bulk: number of unique alignments : 192
2026-05-08T04:14:38.842194Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file24096161d39a42/config_file_2361697.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 @ Fri May  8 00:14:39 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpzTR1Z4/file24096161d39a42/sample1_align2genome.bam
sample2 ->/tmp/RtmpzTR1Z4/file24096161d39a42/sample2_align2genome.bam
sample3 ->/tmp/RtmpzTR1Z4/file24096161d39a42/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri May  8 00:14:59 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:14:59 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpzTR1Z4/file24096161d39a42/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpzTR1Z4/file24096161d39a42/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpzTR1Z4/file24096161d39a42/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:15:21 2026 ----------
2026-05-08T04:15:21.888637Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:15:21.889241Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file24096161d39a42/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-05-08T04:15:21.889265Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:15:21.889273Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:15:21.889376Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:15:21.889392Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-08T04:15:21.899443Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:15:21.899650Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:15:21.899732Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-05-08T04:15:21.899746Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-08T04:15:21.899754Z  INFO oarfish::bulk: number of unique alignments : 208
2026-05-08T04:15:21.900820Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:15:21.910058Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:15:21.910402Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file24096161d39a42/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-05-08T04:15:21.910450Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:15:21.910459Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:15:21.910538Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:15:21.910561Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-08T04:15:21.919513Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:15:21.919745Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:15:21.919821Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-05-08T04:15:21.919829Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-08T04:15:21.919835Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-08T04:15:21.920750Z  INFO oarfish: oarfish completed successfully.
2026-05-08T04:15:21.929981Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:15:21.930349Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file24096161d39a42/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-05-08T04:15:21.930395Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:15:21.930403Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:15:21.930486Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:15:21.930500Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-08T04:15:21.939401Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-08T04:15:21.939611Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-08T04:15:21.939692Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-05-08T04:15:21.939700Z  INFO oarfish::bulk: number of aligned reads : 291
2026-05-08T04:15:21.939706Z  INFO oarfish::bulk: number of unique alignments : 206
2026-05-08T04:15:21.940572Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409613d457563/config_file_2361697.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 @ Fri May  8 00:15:22 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpzTR1Z4/file2409613d457563/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpzTR1Z4/file2409613d457563/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpzTR1Z4/file2409613d457563/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri May  8 00:15:24 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:15:24 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpzTR1Z4/file2409613d457563/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpzTR1Z4/file2409613d457563/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpzTR1Z4/file2409613d457563/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri May  8 00:15:26 2026 ----------
00:15:26 Fri May 08 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/RtmpzTR1Z4/file24096120a277c8/config_file_2361697.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 @ Fri May  8 00:15:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpzTR1Z4/file24096120a277c8/sample1_align2genome.bam
sample2 ->/tmp/RtmpzTR1Z4/file24096120a277c8/sample2_align2genome.bam
sample3 ->/tmp/RtmpzTR1Z4/file24096120a277c8/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri May  8 00:15:47 2026 -------------
Inputs:  ['/tmp/RtmpzTR1Z4/file2409613d457563/sample1_realign2transcript.bam', '/tmp/RtmpzTR1Z4/file2409613d457563/sample2_realign2transcript.bam', '/tmp/RtmpzTR1Z4/file2409613d457563/sample3_realign2transcript.bam'] /tmp/RtmpzTR1Z4/file2409613d457563/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 @ Fri May  8 00:15:48 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpzTR1Z4/file24096120a277c8/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpzTR1Z4/file24096120a277c8/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpzTR1Z4/file24096120a277c8/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:16:09 2026 ----------
00:16:09 Fri May 08 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/RtmpzTR1Z4/file2409612c31c5cc/config_file_2361697.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 @ Fri May  8 00:16:10 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409612c31c5cc/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 @ Fri May  8 00:16:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpzTR1Z4/file2409612c31c5cc/matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409612c31c5cc/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri May  8 00:16:10 2026 ----------------
00:16:10 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409612c31c5cc/align2genome.bam'
Inputs:  ['/tmp/RtmpzTR1Z4/file24096120a277c8/sample1_realign2transcript.bam', '/tmp/RtmpzTR1Z4/file24096120a277c8/sample2_realign2transcript.bam', '/tmp/RtmpzTR1Z4/file24096120a277c8/sample3_realign2transcript.bam'] /tmp/RtmpzTR1Z4/file24096120a277c8/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,  2.42gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.41gene_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, 418392.79Read/s]
-- Running step: isoform_identification @ Fri May  8 00:16:12 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 @ Fri May  8 00:16:22 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/RtmpzTR1Z4/file2409612c31c5cc/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409612c31c5cc/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpzTR1Z4/file2409612c31c5cc/matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409612c31c5cc/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 @ Fri May  8 00:16:22 2026 ----------
2026-05-08T04:16:22.445328Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:16:22.445953Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409612c31c5cc/realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:16:22.446003Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:16:22.446011Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:16:22.446063Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:16:22.446091Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:16:22.451523Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file24096172572046/config_file_2361697.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 @ Fri May  8 00:16:22 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file24096172572046/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 @ Fri May  8 00:16:23 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpzTR1Z4/file24096172572046/matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file24096172572046/align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:16:42 2026 ----------------
00:16:42 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file24096172572046/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.75gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.74gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 413770.03Read/s]
-- Running step: isoform_identification @ Fri May  8 00:16:43 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 @ Fri May  8 00:16:52 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/RtmpzTR1Z4/file24096172572046/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file24096172572046/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpzTR1Z4/file24096172572046/matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file24096172572046/realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:17:11 2026 ----------
2026-05-08T04:17:11.360544Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:17:11.361094Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file24096172572046/realign2transcript.bam, contains 5 reference sequences.
2026-05-08T04:17:11.361164Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:17:11.361173Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:17:11.361237Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:17:11.361260Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-08T04:17:11.366723Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409611987e6bb/config_file_2361697.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 @ Fri May  8 00:17:11 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409611987e6bb/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 @ Fri May  8 00:17:12 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpzTR1Z4/file2409611987e6bb/matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611987e6bb/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 @ Fri May  8 00:17:12 2026 ----------------
00:17:12 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409611987e6bb/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.55gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.54gene_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, 390632.94Read/s]
-- Running step: isoform_identification @ Fri May  8 00:17:13 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 @ Fri May  8 00:17:22 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/RtmpzTR1Z4/file2409611987e6bb/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611987e6bb/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpzTR1Z4/file2409611987e6bb/matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611987e6bb/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 @ Fri May  8 00:17:22 2026 ----------
00:17:22 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409613d9005a4/config_file_2361697.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 @ Fri May  8 00:17:23 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409613d9005a4/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 @ Fri May  8 00:17:23 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409613d9005a4/matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613d9005a4/align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:17:44 2026 ----------------
00:17:44 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409613d9005a4/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,  2.61gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.60gene_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, 398667.78Read/s]
-- Running step: isoform_identification @ Fri May  8 00:17:44 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 @ Fri May  8 00:17:54 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/RtmpzTR1Z4/file2409613d9005a4/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613d9005a4/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409613d9005a4/matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613d9005a4/realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:18:13 2026 ----------
00:18:13 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409613e610d64/config_file_2361697.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 @ Fri May  8 00:18:14 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409613e610d64/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 @ Fri May  8 00:18:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpzTR1Z4/file2409613e610d64/matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e610d64/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri May  8 00:18:14 2026 ----------------
00:18:14 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409613e610d64/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,  2.69gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.69gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 366225.20Read/s]
-- Running step: isoform_identification @ Fri May  8 00:18:15 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:18:15 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/RtmpzTR1Z4/file2409613e610d64/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613e610d64/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpzTR1Z4/file2409613e610d64/matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e610d64/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 @ Fri May  8 00:18:15 2026 ----------
2026-05-08T04:18:15.961177Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:18:15.961650Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409613e610d64/realign2transcript.bam, contains 10 reference sequences.
2026-05-08T04:18:15.961683Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:18:15.961732Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:18:15.961807Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:18:15.961820Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-08T04:18:15.971322Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file24096170acae8f/config_file_2361697.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 @ Fri May  8 00:18:16 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file24096170acae8f/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 @ Fri May  8 00:18:16 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpzTR1Z4/file24096170acae8f/matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file24096170acae8f/align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:18:35 2026 ----------------
00:18:36 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file24096170acae8f/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.52gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.51gene_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, 366634.97Read/s]
-- Running step: isoform_identification @ Fri May  8 00:18:36 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:18:37 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/RtmpzTR1Z4/file24096170acae8f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file24096170acae8f/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpzTR1Z4/file24096170acae8f/matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file24096170acae8f/realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:18:56 2026 ----------
2026-05-08T04:18:56.798104Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:18:56.798609Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file24096170acae8f/realign2transcript.bam, contains 10 reference sequences.
2026-05-08T04:18:56.798635Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:18:56.798644Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:18:56.798725Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:18:56.798770Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-08T04:18:56.806539Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409614c172bae/config_file_2361697.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 @ Fri May  8 00:18:57 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409614c172bae/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 @ Fri May  8 00:18:57 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpzTR1Z4/file2409614c172bae/matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409614c172bae/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 @ Fri May  8 00:18:58 2026 ----------------
00:18:58 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409614c172bae/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.42gene_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, 346728.39Read/s]
-- Running step: isoform_identification @ Fri May  8 00:18:58 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:18:59 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/RtmpzTR1Z4/file2409614c172bae/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409614c172bae/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpzTR1Z4/file2409614c172bae/matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409614c172bae/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 @ Fri May  8 00:18:59 2026 ----------
00:18:59 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409613735d0ac/config_file_2361697.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 @ Fri May  8 00:19:00 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409613735d0ac/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 @ Fri May  8 00:19:00 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409613735d0ac/matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613735d0ac/align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:19:24 2026 ----------------
00:19:24 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409613735d0ac/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,  2.43gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.43gene_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, 374277.56Read/s]
-- Running step: isoform_identification @ Fri May  8 00:19:25 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:19:25 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/RtmpzTR1Z4/file2409613735d0ac/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613735d0ac/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409613735d0ac/matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613735d0ac/realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:19:43 2026 ----------
00:19:43 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409613e2f40f5/config_file_2361697.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 @ Fri May  8 00:19:44 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409613e2f40f5/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/RtmpzTR1Z4/file2409613e2f40f5/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409613e2f40f5/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409613e2f40f5/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/RtmpzTR1Z4/file2409613e2f40f5/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/RtmpzTR1Z4/file2409613e2f40f5/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/RtmpzTR1Z4/file2409613e2f40f5/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/RtmpzTR1Z4/file2409613e2f40f5/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/RtmpzTR1Z4/file2409613e2f40f5/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/RtmpzTR1Z4/file2409613e2f40f5/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 @ Fri May  8 00:19:45 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpzTR1Z4/file2409613e2f40f5/sampleA_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e2f40f5/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri May  8 00:19:49 2026 ----------------
00:19:49 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409613e2f40f5/sampleA_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_align2genome.bam', and
'/tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/RtmpzTR1Z4/file2409613e2f40f5/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, 179369.47Read/s]
parsing /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.27gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 510852.58Read/s]
parsing /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.99gene_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, 563538.45Read/s]
parsing /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.84gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 430662.07Read/s]
-- Running step: isoform_identification @ Fri May  8 00:19:50 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 @ Fri May  8 00:20:14 2026 -------------------
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613e2f40f5/fastq, /tmp/RtmpzTR1Z4/file2409613e2f40f5/fastq/sample1.fq.gz, /tmp/RtmpzTR1Z4/file2409613e2f40f5/fastq/sample2.fq.gz, /tmp/RtmpzTR1Z4/file2409613e2f40f5/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613e2f40f5/sampleA_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613e2f40f5/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpzTR1Z4/file2409613e2f40f5/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e2f40f5/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri May  8 00:20:16 2026 ----------
2026-05-08T04:20:16.323340Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:20:16.323707Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409613e2f40f5/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:20:16.323727Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:20:16.323780Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:20:16.323833Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:20:16.323844Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:20:16.332784Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:20:16.617728Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:20:16.618201Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:20:16.618222Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:20:16.618274Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:20:16.618331Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:20:16.618342Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:20:16.622114Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:20:16.925794Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:20:16.926193Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:20:16.926214Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:20:16.926221Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:20:16.926300Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:20:16.926311Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:20:16.929786Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:20:17.205370Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:20:17.205882Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409613e2f40f5/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:20:17.205902Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:20:17.205910Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:20:17.206000Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:20:17.206012Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:20:17.210069Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409611963114c/config_file_2361697.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 @ Fri May  8 00:20:17 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409611963114c/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/RtmpzTR1Z4/file2409611963114c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409611963114c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409611963114c/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/RtmpzTR1Z4/file2409611963114c/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/RtmpzTR1Z4/file2409611963114c/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/RtmpzTR1Z4/file2409611963114c/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/RtmpzTR1Z4/file2409611963114c/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/RtmpzTR1Z4/file2409611963114c/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/RtmpzTR1Z4/file2409611963114c/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 @ Fri May  8 00:20:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409611963114c/sampleA_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611963114c/sampleA_align2genome.bam
/tmp/RtmpzTR1Z4/file2409611963114c/sample1_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611963114c/sample1_align2genome.bam
/tmp/RtmpzTR1Z4/file2409611963114c/sample2_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611963114c/sample2_align2genome.bam
/tmp/RtmpzTR1Z4/file2409611963114c/sample3_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611963114c/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:20:40 2026 ----------------
00:20:40 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409611963114c/sampleA_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409611963114c/sample1_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409611963114c/sample2_align2genome.bam', and
'/tmp/RtmpzTR1Z4/file2409611963114c/sample3_align2genome.bam'
parsing /tmp/RtmpzTR1Z4/file2409611963114c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.03gene_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, 153003.08Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611963114c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.43gene_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, 557367.78Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611963114c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 30.50gene_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, 495148.51Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611963114c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.57gene_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, 407087.51Read/s]
-- Running step: isoform_identification @ Fri May  8 00:20:42 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  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 @ Fri May  8 00:21:06 2026 -------------------
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611963114c/fastq, /tmp/RtmpzTR1Z4/file2409611963114c/fastq/sample1.fq.gz, /tmp/RtmpzTR1Z4/file2409611963114c/fastq/sample2.fq.gz, /tmp/RtmpzTR1Z4/file2409611963114c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611963114c/sampleA_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611963114c/sample1_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611963114c/sample2_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611963114c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611963114c/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611963114c/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611963114c/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611963114c/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409611963114c/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611963114c/sampleA_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409611963114c/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611963114c/sample1_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409611963114c/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611963114c/sample2_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409611963114c/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611963114c/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:21:27 2026 ----------
2026-05-08T04:21:27.030136Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:21:27.030495Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611963114c/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:21:27.030555Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:21:27.030564Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:21:27.030622Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:21:27.030635Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:21:27.039829Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:21:27.396110Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:21:27.396474Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611963114c/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:21:27.396496Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:21:27.396552Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:21:27.396606Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:21:27.396617Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:21:27.400658Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:21:27.717062Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:21:27.717435Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611963114c/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:21:27.717459Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:21:27.717506Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:21:27.717560Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:21:27.717572Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:21:27.721480Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:21:28.047409Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:21:28.047767Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611963114c/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-08T04:21:28.047788Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:21:28.047796Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:21:28.047896Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:21:28.047908Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-08T04:21:28.052013Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file240961775121e5/config_file_2361697.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 @ Fri May  8 00:21:28 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file240961775121e5/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/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 @ Fri May  8 00:21:29 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpzTR1Z4/file240961775121e5/sampleA_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/sample1_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/sample2_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/sample3_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file240961775121e5/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 @ Fri May  8 00:21:33 2026 ----------------
00:21:33 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file240961775121e5/sampleA_align2genome.bam',
'/tmp/RtmpzTR1Z4/file240961775121e5/sample1_align2genome.bam',
'/tmp/RtmpzTR1Z4/file240961775121e5/sample2_align2genome.bam', and
'/tmp/RtmpzTR1Z4/file240961775121e5/sample3_align2genome.bam'
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.70gene_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, 150255.93Read/s]
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.61gene_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, 538587.50Read/s]
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.35gene_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, 506313.86Read/s]
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.16gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 400755.21Read/s]
-- Running step: isoform_identification @ Fri May  8 00:21:34 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 @ Fri May  8 00:21:58 2026 -------------------
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file240961775121e5/fastq, /tmp/RtmpzTR1Z4/file240961775121e5/fastq/sample1.fq.gz, /tmp/RtmpzTR1Z4/file240961775121e5/fastq/sample2.fq.gz, /tmp/RtmpzTR1Z4/file240961775121e5/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file240961775121e5/sampleA_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file240961775121e5/sample1_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file240961775121e5/sample2_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file240961775121e5/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file240961775121e5/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file240961775121e5/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file240961775121e5/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file240961775121e5/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpzTR1Z4/file240961775121e5/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file240961775121e5/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/RtmpzTR1Z4/file240961775121e5/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file240961775121e5/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 @ Fri May  8 00:21:59 2026 ----------
00:21:59 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sampleA_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file240961775121e5/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample1_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file240961775121e5/sample1_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample2_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file240961775121e5/sample2_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample3_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file240961775121e5/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file240961775121e5/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409615ef04adf/config_file_2361697.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 @ Fri May  8 00:22:02 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409615ef04adf/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/RtmpzTR1Z4/file2409615ef04adf/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409615ef04adf/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409615ef04adf/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/RtmpzTR1Z4/file2409615ef04adf/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/RtmpzTR1Z4/file2409615ef04adf/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/RtmpzTR1Z4/file2409615ef04adf/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/RtmpzTR1Z4/file2409615ef04adf/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/RtmpzTR1Z4/file2409615ef04adf/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/RtmpzTR1Z4/file2409615ef04adf/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 @ Fri May  8 00:22:03 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_align2genome.bam
/tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_align2genome.bam
/tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_align2genome.bam
/tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:22:25 2026 ----------------
00:22:25 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_align2genome.bam', and
'/tmp/RtmpzTR1Z4/file2409615ef04adf/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/RtmpzTR1Z4/file2409615ef04adf/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.57gene_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, 168235.14Read/s]
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.88gene_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, 359556.97Read/s]
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.88gene_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, 510927.25Read/s]
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.73gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 394929.00Read/s]
-- Running step: isoform_identification @ Fri May  8 00:22:27 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)
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 @ Fri May  8 00:22:50 2026 -------------------
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409615ef04adf/fastq, /tmp/RtmpzTR1Z4/file2409615ef04adf/fastq/sample1.fq.gz, /tmp/RtmpzTR1Z4/file2409615ef04adf/fastq/sample2.fq.gz, /tmp/RtmpzTR1Z4/file2409615ef04adf/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:23:11 2026 ----------
00:23:11 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409615ef04adf/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409615ef04adf/sample1_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409615ef04adf/sample2_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409615ef04adf/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409611485fa3b/config_file_2361697.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 @ Fri May  8 00:23:13 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409611485fa3b/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/RtmpzTR1Z4/file2409611485fa3b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409611485fa3b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409611485fa3b/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/RtmpzTR1Z4/file2409611485fa3b/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/RtmpzTR1Z4/file2409611485fa3b/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/RtmpzTR1Z4/file2409611485fa3b/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/RtmpzTR1Z4/file2409611485fa3b/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/RtmpzTR1Z4/file2409611485fa3b/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/RtmpzTR1Z4/file2409611485fa3b/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 @ Fri May  8 00:23:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpzTR1Z4/file2409611485fa3b/sampleA_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611485fa3b/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpzTR1Z4/file2409611485fa3b/sample3_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611485fa3b/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri May  8 00:23:18 2026 ----------------
00:23:18 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409611485fa3b/sampleA_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_align2genome.bam', and
'/tmp/RtmpzTR1Z4/file2409611485fa3b/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/RtmpzTR1Z4/file2409611485fa3b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.91gene_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, 162597.65Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.42gene_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, 496062.07Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.92gene_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, 467801.03Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611485fa3b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.48gene_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, 407403.84Read/s]
-- Running step: isoform_identification @ Fri May  8 00:23:19 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:23:20 2026 -------------------
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611485fa3b/fastq, /tmp/RtmpzTR1Z4/file2409611485fa3b/fastq/sample1.fq.gz, /tmp/RtmpzTR1Z4/file2409611485fa3b/fastq/sample2.fq.gz, /tmp/RtmpzTR1Z4/file2409611485fa3b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611485fa3b/sampleA_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611485fa3b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611485fa3b/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611485fa3b/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpzTR1Z4/file2409611485fa3b/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611485fa3b/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpzTR1Z4/file2409611485fa3b/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409611485fa3b/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri May  8 00:23:27 2026 ----------
2026-05-08T04:23:27.646368Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:23:27.646765Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611485fa3b/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-05-08T04:23:27.646791Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:23:27.646801Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:23:27.647166Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:23:27.647191Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-08T04:23:27.686327Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:23:28.277504Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:23:28.277949Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611485fa3b/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-05-08T04:23:28.277974Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:23:28.277983Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:23:28.278096Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:23:28.278116Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-08T04:23:28.291610Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:23:28.819058Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:23:28.819553Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611485fa3b/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-05-08T04:23:28.819578Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:23:28.819588Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:23:28.819711Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:23:28.819733Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-08T04:23:28.835309Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:23:29.360147Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:23:29.360536Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611485fa3b/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-05-08T04:23:29.360561Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:23:29.360571Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:23:29.360695Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:23:29.360715Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-08T04:23:29.376702Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409611278f81f/config_file_2361697.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 @ Fri May  8 00:23:30 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409611278f81f/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/RtmpzTR1Z4/file2409611278f81f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409611278f81f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409611278f81f/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/RtmpzTR1Z4/file2409611278f81f/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/RtmpzTR1Z4/file2409611278f81f/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/RtmpzTR1Z4/file2409611278f81f/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/RtmpzTR1Z4/file2409611278f81f/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/RtmpzTR1Z4/file2409611278f81f/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/RtmpzTR1Z4/file2409611278f81f/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 @ Fri May  8 00:23:31 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_align2genome.bam
/tmp/RtmpzTR1Z4/file2409611278f81f/sample1_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611278f81f/sample1_align2genome.bam
/tmp/RtmpzTR1Z4/file2409611278f81f/sample2_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611278f81f/sample2_align2genome.bam
/tmp/RtmpzTR1Z4/file2409611278f81f/sample3_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611278f81f/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:23:54 2026 ----------------
00:23:54 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409611278f81f/sample1_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409611278f81f/sample2_align2genome.bam', and
'/tmp/RtmpzTR1Z4/file2409611278f81f/sample3_align2genome.bam'
parsing /tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 11.13gene_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, 143025.34Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611278f81f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.43gene_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, 472778.75Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611278f81f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.82gene_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, 478779.96Read/s]
parsing /tmp/RtmpzTR1Z4/file2409611278f81f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.57gene_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, 413264.49Read/s]
-- Running step: isoform_identification @ Fri May  8 00:23:55 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:23:56 2026 -------------------
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611278f81f/fastq, /tmp/RtmpzTR1Z4/file2409611278f81f/fastq/sample1.fq.gz, /tmp/RtmpzTR1Z4/file2409611278f81f/fastq/sample2.fq.gz, /tmp/RtmpzTR1Z4/file2409611278f81f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611278f81f/sample1_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611278f81f/sample2_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409611278f81f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611278f81f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611278f81f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409611278f81f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409611278f81f/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611278f81f/sample1_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409611278f81f/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611278f81f/sample2_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409611278f81f/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409611278f81f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:24:26 2026 ----------
2026-05-08T04:24:26.318288Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:24:26.318777Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611278f81f/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-05-08T04:24:26.318805Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:24:26.318815Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:24:26.318939Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:24:26.318960Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-08T04:24:26.358331Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:24:27.000296Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:24:27.000930Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611278f81f/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-05-08T04:24:27.000957Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:24:27.000966Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:24:27.001082Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:24:27.001102Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-08T04:24:27.014779Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:24:27.597637Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:24:27.598137Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611278f81f/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-05-08T04:24:27.598163Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:24:27.598178Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:24:27.598300Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:24:27.598321Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-08T04:24:27.612282Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-08T04:24:28.223948Z  INFO oarfish: setting user-provided filter parameters.
2026-05-08T04:24:28.224347Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpzTR1Z4/file2409611278f81f/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-05-08T04:24:28.224374Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-08T04:24:28.224384Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-08T04:24:28.224506Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-08T04:24:28.224527Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-08T04:24:28.241012Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409615dbdbd42/config_file_2361697.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 @ Fri May  8 00:24:29 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409615dbdbd42/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/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 @ Fri May  8 00:24:30 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/sample1_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/sample2_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/sample3_matched_reads.fastq.gz -> /tmp/RtmpzTR1Z4/file2409615dbdbd42/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 @ Fri May  8 00:24:33 2026 ----------------
00:24:33 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409615dbdbd42/sample1_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409615dbdbd42/sample2_align2genome.bam', and
'/tmp/RtmpzTR1Z4/file2409615dbdbd42/sample3_align2genome.bam'
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.85gene_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, 175558.53Read/s]
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.32gene_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, 527214.04Read/s]
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.45gene_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, 534333.47Read/s]
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.78gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 391303.50Read/s]
-- Running step: isoform_identification @ Fri May  8 00:24:35 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:24:35 2026 -------------------
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409615dbdbd42/fastq, /tmp/RtmpzTR1Z4/file2409615dbdbd42/fastq/sample1.fq.gz, /tmp/RtmpzTR1Z4/file2409615dbdbd42/fastq/sample2.fq.gz, /tmp/RtmpzTR1Z4/file2409615dbdbd42/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample1_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample2_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409615dbdbd42/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/RtmpzTR1Z4/file2409615dbdbd42/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpzTR1Z4/file2409615dbdbd42/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 @ Fri May  8 00:24:38 2026 ----------
00:24:38 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409615dbdbd42/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample1_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409615dbdbd42/sample1_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample2_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409615dbdbd42/sample2_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample3_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409615dbdbd42/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409615dbdbd42/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpzTR1Z4/file2409613bb777e4/config_file_2361697.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 @ Fri May  8 00:24:41 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpzTR1Z4/file2409613bb777e4/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/RtmpzTR1Z4/file2409613bb777e4/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409613bb777e4/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpzTR1Z4/file2409613bb777e4/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/RtmpzTR1Z4/file2409613bb777e4/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/RtmpzTR1Z4/file2409613bb777e4/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/RtmpzTR1Z4/file2409613bb777e4/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/RtmpzTR1Z4/file2409613bb777e4/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/RtmpzTR1Z4/file2409613bb777e4/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/RtmpzTR1Z4/file2409613bb777e4/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 @ Fri May  8 00:24:42 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_align2genome.bam
/tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_align2genome.bam
/tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_align2genome.bam
/tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_matched_reads.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri May  8 00:25:05 2026 ----------------
00:25:05 Fri May 08 2026 quantify genes 
Using BAM(s): '/tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_align2genome.bam',
'/tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_align2genome.bam', and
'/tmp/RtmpzTR1Z4/file2409613bb777e4/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/RtmpzTR1Z4/file2409613bb777e4/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.57gene_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, 151842.10Read/s]
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.61gene_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, 496908.35Read/s]
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.64gene_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, 524419.10Read/s]
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.82gene_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, 400464.41Read/s]
-- Running step: isoform_identification @ Fri May  8 00:25:06 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri May  8 00:25:07 2026 -------------------
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613bb777e4/fastq, /tmp/RtmpzTR1Z4/file2409613bb777e4/fastq/sample1.fq.gz, /tmp/RtmpzTR1Z4/file2409613bb777e4/fastq/sample2.fq.gz, /tmp/RtmpzTR1Z4/file2409613bb777e4/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_matched_reads.fastq.gz, /tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_realign2transcript.bam
/tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri May  8 00:25:29 2026 ----------
00:25:29 Fri May 08 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409613bb777e4/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409613bb777e4/sample1_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409613bb777e4/sample2_realign2transcript.bamdone
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_realign2transcript.bam...
parsing /tmp/RtmpzTR1Z4/file2409613bb777e4/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpzTR1Z4/file2409613bb777e4/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 ...

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

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

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |=======================                                               |  33%
  |                                                                            
  |===============================================                       |  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 
829.312  56.174 869.781