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###
### Running command:
###
###   /home/biocbuild/bbs-3.24-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.24-bioc/R/site-library --timings FLAMES_2.7.0.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.24-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: 2026-05-25 04:01:25 UTC
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.7.0’
* 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-3.24-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 13.3Mb
  sub-directories of 1Mb or more:
    bin    8.3Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
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-3.24-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
find_variants                21.950  1.752  22.631
plot_isoform_reduced_dim     20.931  0.839  21.770
blaze                         6.304 14.536  15.742
bulk_long_pipeline            6.826 13.778   2.494
sc_plot_genotype             11.762  2.040  11.808
sc_long_multisample_pipeline  8.532  5.131   8.352
sc_DTU_analysis               8.993  1.646   9.413
MultiSampleSCPipeline         9.867  0.706  10.803
create_se_from_dir            5.351  0.170   5.507
create_sce_from_dir           3.555  1.782   3.524
plot_durations                5.110  0.115   5.211
* 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-3.24-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.24-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
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* installing to library ‘/home/biocbuild/bbs-3.24-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.7.0’
** 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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-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-3.24-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-3.24-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.24-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-3.24-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-3.24-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.24-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)
   Compiling libc v0.2.186
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   Compiling proc-macro-error-attr2 v2.0.0
   Compiling crossbeam-deque v0.8.6
   Compiling jobserver v0.1.34
   Compiling cc v1.2.62
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   Compiling getrandom v0.2.17
   Compiling num_cpus v1.17.0
   Compiling console v0.15.11
   Compiling block-buffer v0.10.4
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   Compiling rand_core v0.6.4
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   Compiling tempfile v3.27.0
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   Compiling ndarray v0.16.1
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   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 sha2 v0.10.9
   Compiling csv v1.4.0
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   Compiling num-rational v0.4.2
   Compiling proc-macro-error2 v2.0.1
   Compiling num v0.4.3
   Compiling bytemuck_derive v1.10.2
   Compiling futures-macro v0.3.32
   Compiling serde_derive v1.0.228
   Compiling async-stream-impl v0.3.6
   Compiling tracing-attributes v0.1.31
   Compiling async-trait v0.1.89
   Compiling thiserror-impl v1.0.69
   Compiling strum_macros v0.26.4
   Compiling typed-builder-macro v0.21.2
   Compiling clap_derive v4.6.1
   Compiling derive-new v0.6.0
   Compiling tabled_derive v0.10.0
   Compiling async-stream v0.3.6
   Compiling futures-util v0.3.32
   Compiling ppv-lite86 v0.2.21
   Compiling bytemuck v1.25.0
   Compiling hashbrown v0.14.5
   Compiling typed-builder v0.21.2
   Compiling tabled v0.18.0
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   Compiling safe_arch v0.7.4
   Compiling rand_chacha v0.3.1
   Compiling rand_chacha v0.9.0
   Compiling bstr v1.12.1
   Compiling matchers v0.2.0
   Compiling regex v1.12.3
   Compiling bzip2 v0.4.4
   Compiling tracing-subscriber v0.3.23
   Compiling rand v0.8.6
   Compiling rand v0.9.4
   Compiling wide v0.7.33
   Compiling noodles-core v0.17.0
   Compiling noodles-csi v0.46.0
   Compiling clap v4.6.1
   Compiling rand_distr v0.4.3
   Compiling noodles-sam v0.74.0
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   Compiling simba v0.9.1
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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 51.47s
  Installing /home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/cargo_temp/bin/oarfish
   Installed package `oarfish v0.8.0` (executable `oarfish`)
warning: be sure to add `/home/biocbuild/bbs-3.24-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-3.24-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/RtmpVKt6Bd/filec9f501224720e/config_file_827216.json
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f501224720e/config_file_827216.json
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f501224720e/config_file_827216.json
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f507640e03a/config_file_827216.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f505d468369/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-3.24-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/RtmpVKt6Bd/filec9f502de579b8/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-3.24-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/RtmpVKt6Bd/filec9f502de579b8/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/RtmpVKt6Bd/filec9f5032fc722a/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5032fc722a/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5032fc722a/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5032fc722a/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/RtmpVKt6Bd/filec9f5049e37afc/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-3.24-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/RtmpVKt6Bd/filec9f5037eafed6/config_file_827216.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 @ Mon May 25 00:10:31 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon May 25 00:10:33 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 @ Mon May 25 00:10:59 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon May 25 00:11:00 2026 ----------
2026-05-25T04:11:00.141276Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:11:00.141828Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-25T04:11:00.141845Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:11:00.141850Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:11:00.141925Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:11:00.141931Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-25T04:11:00.146181Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-25T04:11:00.146388Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:11:00.146418Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-25T04:11:00.146421Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-25T04:11:00.146424Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-25T04:11:00.147265Z  INFO oarfish: oarfish completed successfully.
2026-05-25T04:11:00.154519Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:11:00.154866Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-25T04:11:00.154874Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:11:00.154877Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:11:00.154933Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:11:00.154938Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-25T04:11:00.158980Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-25T04:11:00.159167Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:11:00.159195Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-05-25T04:11:00.159198Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-25T04:11:00.159200Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-25T04:11:00.160036Z  INFO oarfish: oarfish completed successfully.
2026-05-25T04:11:00.167058Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:11:00.167409Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f5037eafed6/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-25T04:11:00.167421Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:11:00.167425Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:11:00.167484Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:11:00.167490Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-25T04:11:00.171544Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-25T04:11:00.171720Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:11:00.171755Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-05-25T04:11:00.171757Z  INFO oarfish::bulk: number of aligned reads : 284
2026-05-25T04:11:00.171760Z  INFO oarfish::bulk: number of unique alignments : 237
2026-05-25T04:11:00.172598Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f50ac7f269/config_file_827216.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 @ Mon May 25 00:11:00 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpVKt6Bd/filec9f50ac7f269/sample1_align2genome.bam
sample2 ->/tmp/RtmpVKt6Bd/filec9f50ac7f269/sample2_align2genome.bam
sample3 ->/tmp/RtmpVKt6Bd/filec9f50ac7f269/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon May 25 00:11:21 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon May 25 00:11:43 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpVKt6Bd/filec9f50ac7f269/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpVKt6Bd/filec9f50ac7f269/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpVKt6Bd/filec9f50ac7f269/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:12:02 2026 ----------
2026-05-25T04:12:03.015500Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:12:03.015961Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50ac7f269/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-25T04:12:03.015981Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:12:03.015985Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:12:03.016043Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:12:03.016050Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-25T04:12:03.020262Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-25T04:12:03.020456Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:12:03.020488Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-25T04:12:03.020491Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-25T04:12:03.020494Z  INFO oarfish::bulk: number of unique alignments : 238
2026-05-25T04:12:03.021324Z  INFO oarfish: oarfish completed successfully.
2026-05-25T04:12:03.030027Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:12:03.030406Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50ac7f269/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-25T04:12:03.030418Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:12:03.030422Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:12:03.030499Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:12:03.030506Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-25T04:12:03.034482Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-25T04:12:03.034697Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:12:03.034720Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-05-25T04:12:03.034723Z  INFO oarfish::bulk: number of aligned reads : 282
2026-05-25T04:12:03.034725Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-25T04:12:03.035584Z  INFO oarfish: oarfish completed successfully.
2026-05-25T04:12:03.044635Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:12:03.045173Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50ac7f269/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-25T04:12:03.045181Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:12:03.045185Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:12:03.045242Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:12:03.045248Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-25T04:12:03.049320Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-25T04:12:03.049529Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:12:03.049562Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-05-25T04:12:03.049565Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-25T04:12:03.049568Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-25T04:12:03.050446Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f5043142ce5/config_file_827216.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 @ Mon May 25 00:12:03 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpVKt6Bd/filec9f5043142ce5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpVKt6Bd/filec9f5043142ce5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpVKt6Bd/filec9f5043142ce5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon May 25 00:12:05 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 @ Mon May 25 00:12:25 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpVKt6Bd/filec9f5043142ce5/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpVKt6Bd/filec9f5043142ce5/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpVKt6Bd/filec9f5043142ce5/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon May 25 00:12:26 2026 ----------
00:12:26 Mon May 25 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/RtmpVKt6Bd/filec9f501b166a45/config_file_827216.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 @ Mon May 25 00:12:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpVKt6Bd/filec9f501b166a45/sample1_align2genome.bam
sample2 ->/tmp/RtmpVKt6Bd/filec9f501b166a45/sample2_align2genome.bam
sample3 ->/tmp/RtmpVKt6Bd/filec9f501b166a45/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon May 25 00:12:49 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 @ Mon May 25 00:13:09 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpVKt6Bd/filec9f501b166a45/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpVKt6Bd/filec9f501b166a45/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpVKt6Bd/filec9f501b166a45/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:13:28 2026 ----------
00:13:28 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpVKt6Bd/filec9f5043142ce5/sample1_realign2transcript.bam', '/tmp/RtmpVKt6Bd/filec9f5043142ce5/sample2_realign2transcript.bam', '/tmp/RtmpVKt6Bd/filec9f5043142ce5/sample3_realign2transcript.bam'] /tmp/RtmpVKt6Bd/filec9f5043142ce5/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 866, 'not_enough_coverage': 30, 'unmapped': 4})
Inputs:  ['/tmp/RtmpVKt6Bd/filec9f501b166a45/sample1_realign2transcript.bam', '/tmp/RtmpVKt6Bd/filec9f501b166a45/sample2_realign2transcript.bam', '/tmp/RtmpVKt6Bd/filec9f501b166a45/sample3_realign2transcript.bam'] /tmp/RtmpVKt6Bd/filec9f501b166a45/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f505762f410/config_file_827216.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 @ Mon May 25 00:13:29 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpVKt6Bd/filec9f505762f410/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpVKt6Bd/filec9f505762f410/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpVKt6Bd/filec9f505762f410/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon May 25 00:13:30 2026 -------------
	Counter({'counted_reads': 863, 'not_enough_coverage': 32, 'unmapped': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:13:31 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpVKt6Bd/filec9f505762f410/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpVKt6Bd/filec9f505762f410/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpVKt6Bd/filec9f505762f410/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon May 25 00:13:34 2026 ----------
2026-05-25T04:13:34.198928Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:13:34.199597Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f505762f410/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-05-25T04:13:34.199611Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:13:34.199614Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:13:34.199716Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:13:34.199727Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-25T04:13:34.210341Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-25T04:13:34.210557Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:13:34.210590Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-05-25T04:13:34.210593Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-25T04:13:34.210595Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-25T04:13:34.211504Z  INFO oarfish: oarfish completed successfully.
2026-05-25T04:13:34.219543Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:13:34.219919Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f505762f410/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-05-25T04:13:34.219927Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:13:34.219931Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:13:34.220025Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:13:34.220034Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-25T04:13:34.230337Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-25T04:13:34.230584Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:13:34.230628Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-05-25T04:13:34.230631Z  INFO oarfish::bulk: number of aligned reads : 292
2026-05-25T04:13:34.230633Z  INFO oarfish::bulk: number of unique alignments : 190
2026-05-25T04:13:34.231535Z  INFO oarfish: oarfish completed successfully.
2026-05-25T04:13:34.239632Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:13:34.240008Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f505762f410/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-05-25T04:13:34.240016Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:13:34.240019Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:13:34.240111Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:13:34.240121Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-25T04:13:34.251312Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-25T04:13:34.251577Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:13:34.251611Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-05-25T04:13:34.251614Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-25T04:13:34.251616Z  INFO oarfish::bulk: number of unique alignments : 192
2026-05-25T04:13:34.252510Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f506837febd/config_file_827216.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 @ Mon May 25 00:13:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpVKt6Bd/filec9f506837febd/sample1_align2genome.bam
sample2 ->/tmp/RtmpVKt6Bd/filec9f506837febd/sample2_align2genome.bam
sample3 ->/tmp/RtmpVKt6Bd/filec9f506837febd/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon May 25 00:13:55 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:13:55 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpVKt6Bd/filec9f506837febd/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpVKt6Bd/filec9f506837febd/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpVKt6Bd/filec9f506837febd/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:14:16 2026 ----------
2026-05-25T04:14:16.494772Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:14:16.495395Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f506837febd/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-05-25T04:14:16.495407Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:14:16.495411Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:14:16.495493Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:14:16.495504Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-25T04:14:16.505207Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-25T04:14:16.505412Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:14:16.505444Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-05-25T04:14:16.505448Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-25T04:14:16.505451Z  INFO oarfish::bulk: number of unique alignments : 208
2026-05-25T04:14:16.506325Z  INFO oarfish: oarfish completed successfully.
2026-05-25T04:14:16.517195Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:14:16.517651Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f506837febd/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-05-25T04:14:16.517664Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:14:16.517667Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:14:16.517758Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:14:16.517768Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-25T04:14:16.527204Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-25T04:14:16.527406Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:14:16.527445Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-05-25T04:14:16.527448Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-25T04:14:16.527450Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-25T04:14:16.528333Z  INFO oarfish: oarfish completed successfully.
2026-05-25T04:14:16.538945Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:14:16.539355Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f506837febd/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-05-25T04:14:16.539362Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:14:16.539365Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:14:16.539455Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:14:16.539466Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-25T04:14:16.548572Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-25T04:14:16.548750Z  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   │
╰─────────────────────────────────┴───────╯

2026-05-25T04:14:16.548778Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-05-25T04:14:16.548781Z  INFO oarfish::bulk: number of aligned reads : 291
2026-05-25T04:14:16.548783Z  INFO oarfish::bulk: number of unique alignments : 206
2026-05-25T04:14:16.549641Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f5045ea7802/config_file_827216.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 @ Mon May 25 00:14:16 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpVKt6Bd/filec9f5045ea7802/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpVKt6Bd/filec9f5045ea7802/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpVKt6Bd/filec9f5045ea7802/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon May 25 00:14:18 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:14:19 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpVKt6Bd/filec9f5045ea7802/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpVKt6Bd/filec9f5045ea7802/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpVKt6Bd/filec9f5045ea7802/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon May 25 00:14:20 2026 ----------
00:14:20 Mon May 25 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/RtmpVKt6Bd/filec9f506d8323d5/config_file_827216.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 @ Mon May 25 00:14:21 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample1_align2genome.bam
sample2 ->/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample2_align2genome.bam
sample3 ->/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon May 25 00:14:45 2026 -------------
Inputs:  ['/tmp/RtmpVKt6Bd/filec9f5045ea7802/sample1_realign2transcript.bam', '/tmp/RtmpVKt6Bd/filec9f5045ea7802/sample2_realign2transcript.bam', '/tmp/RtmpVKt6Bd/filec9f5045ea7802/sample3_realign2transcript.bam'] /tmp/RtmpVKt6Bd/filec9f5045ea7802/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 @ Mon May 25 00:14:46 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:15:05 2026 ----------
00:15:05 Mon May 25 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/RtmpVKt6Bd/filec9f503c1446e2/config_file_827216.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 @ Mon May 25 00:15:06 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f503c1446e2/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-3.24-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 @ Mon May 25 00:15:06 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpVKt6Bd/filec9f503c1446e2/matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f503c1446e2/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon May 25 00:15:07 2026 ----------------
00:15:07 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f503c1446e2/align2genome.bam'
Inputs:  ['/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample1_realign2transcript.bam', '/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample2_realign2transcript.bam', '/tmp/RtmpVKt6Bd/filec9f506d8323d5/sample3_realign2transcript.bam'] /tmp/RtmpVKt6Bd/filec9f506d8323d5/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,  2.70gene_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, 435418.99Read/s]
-- Running step: isoform_identification @ Mon May 25 00:15:08 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 @ Mon May 25 00:15:17 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f503c1446e2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f503c1446e2/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpVKt6Bd/filec9f503c1446e2/matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f503c1446e2/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 @ Mon May 25 00:15:17 2026 ----------
2026-05-25T04:15:17.992301Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:15:17.992758Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f503c1446e2/realign2transcript.bam, contains 5 reference sequences.
2026-05-25T04:15:17.992782Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:15:17.992785Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:15:17.992848Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:15:17.992854Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-25T04:15:17.999244Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f507d05f3ec/config_file_827216.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 @ Mon May 25 00:15:18 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f507d05f3ec/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-3.24-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 @ Mon May 25 00:15:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f507d05f3ec/matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f507d05f3ec/align2genome.bam
-- Running step: gene_quantification @ Mon May 25 00:15:38 2026 ----------------
00:15:38 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f507d05f3ec/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.90gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.90gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 411884.67Read/s]
-- Running step: isoform_identification @ Mon May 25 00:15:38 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 @ Mon May 25 00:15:48 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f507d05f3ec/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f507d05f3ec/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f507d05f3ec/matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f507d05f3ec/realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:16:08 2026 ----------
2026-05-25T04:16:08.473436Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:16:08.473979Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f507d05f3ec/realign2transcript.bam, contains 5 reference sequences.
2026-05-25T04:16:08.473989Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:16:08.473993Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:16:08.474053Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:16:08.474058Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-25T04:16:08.480181Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f507269aeeb/config_file_827216.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 @ Mon May 25 00:16:09 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f507269aeeb/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-3.24-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 @ Mon May 25 00:16:09 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpVKt6Bd/filec9f507269aeeb/matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507269aeeb/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 @ Mon May 25 00:16:09 2026 ----------------
00:16:09 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f507269aeeb/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.71gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.71gene_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, 378055.96Read/s]
-- Running step: isoform_identification @ Mon May 25 00:16:10 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 @ Mon May 25 00:16:19 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f507269aeeb/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f507269aeeb/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpVKt6Bd/filec9f507269aeeb/matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507269aeeb/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 @ Mon May 25 00:16:19 2026 ----------
00:16:19 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f504e597386/config_file_827216.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 @ Mon May 25 00:16:20 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f504e597386/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-3.24-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 @ Mon May 25 00:16:20 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f504e597386/matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f504e597386/align2genome.bam
-- Running step: gene_quantification @ Mon May 25 00:16:38 2026 ----------------
00:16:38 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f504e597386/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,  2.84gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.84gene_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, 450051.93Read/s]
-- Running step: isoform_identification @ Mon May 25 00:16:39 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 @ Mon May 25 00:16:48 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f504e597386/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f504e597386/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f504e597386/matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f504e597386/realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:17:06 2026 ----------
00:17:06 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f504cfd0952/config_file_827216.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 @ Mon May 25 00:17:07 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f504cfd0952/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-3.24-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 @ Mon May 25 00:17:07 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpVKt6Bd/filec9f504cfd0952/matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f504cfd0952/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon May 25 00:17:08 2026 ----------------
00:17:08 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f504cfd0952/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,  2.71gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.71gene_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, 374558.31Read/s]
-- Running step: isoform_identification @ Mon May 25 00:17:08 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:17:09 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f504cfd0952/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f504cfd0952/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpVKt6Bd/filec9f504cfd0952/matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f504cfd0952/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 @ Mon May 25 00:17:09 2026 ----------
2026-05-25T04:17:09.353467Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:17:09.353935Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f504cfd0952/realign2transcript.bam, contains 10 reference sequences.
2026-05-25T04:17:09.353945Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:17:09.353947Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:17:09.354028Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:17:09.354036Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-25T04:17:09.363906Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f5031566f86/config_file_827216.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 @ Mon May 25 00:17:10 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f5031566f86/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-3.24-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 @ Mon May 25 00:17:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f5031566f86/matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5031566f86/align2genome.bam
-- Running step: gene_quantification @ Mon May 25 00:17:29 2026 ----------------
00:17:29 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f5031566f86/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.68gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.67gene_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, 373703.98Read/s]
-- Running step: isoform_identification @ Mon May 25 00:17:30 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:17:30 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5031566f86/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5031566f86/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f5031566f86/matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5031566f86/realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:17:48 2026 ----------
2026-05-25T04:17:48.617323Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:17:48.617768Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f5031566f86/realign2transcript.bam, contains 10 reference sequences.
2026-05-25T04:17:48.617791Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:17:48.617794Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:17:48.617864Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:17:48.617871Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-25T04:17:48.627476Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f506e283677/config_file_827216.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 @ Mon May 25 00:17:49 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f506e283677/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-3.24-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 @ Mon May 25 00:17:49 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpVKt6Bd/filec9f506e283677/matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f506e283677/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 @ Mon May 25 00:17:49 2026 ----------------
00:17:49 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f506e283677/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.56gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.55gene_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, 375752.88Read/s]
-- Running step: isoform_identification @ Mon May 25 00:17:50 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:17:51 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f506e283677/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f506e283677/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpVKt6Bd/filec9f506e283677/matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f506e283677/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 @ Mon May 25 00:17:51 2026 ----------
00:17:51 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f506cb1f179/config_file_827216.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 @ Mon May 25 00:17:52 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f506cb1f179/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-3.24-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 @ Mon May 25 00:17:52 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f506cb1f179/matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f506cb1f179/align2genome.bam
-- Running step: gene_quantification @ Mon May 25 00:18:12 2026 ----------------
00:18:12 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f506cb1f179/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,  2.63gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.62gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 389197.54Read/s]
-- Running step: isoform_identification @ Mon May 25 00:18:12 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:18:13 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f506cb1f179/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f506cb1f179/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f506cb1f179/matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f506cb1f179/realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:18:31 2026 ----------
00:18:31 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f50515d9087/config_file_827216.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 @ Mon May 25 00:18:32 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f50515d9087/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/RtmpVKt6Bd/filec9f50515d9087/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f50515d9087/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f50515d9087/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/RtmpVKt6Bd/filec9f50515d9087/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/RtmpVKt6Bd/filec9f50515d9087/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/RtmpVKt6Bd/filec9f50515d9087/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/RtmpVKt6Bd/filec9f50515d9087/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/RtmpVKt6Bd/filec9f50515d9087/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/RtmpVKt6Bd/filec9f50515d9087/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 @ Mon May 25 00:18:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon May 25 00:18:37 2026 ----------------
00:18:37 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_align2genome.bam', and
'/tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 12.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 180770.27Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 556362.29Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.69gene_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, 574467.76Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 450884.07Read/s]
-- Running step: isoform_identification @ Mon May 25 00:18:38 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 @ Mon May 25 00:19:01 2026 -------------------
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50515d9087/fastq, /tmp/RtmpVKt6Bd/filec9f50515d9087/fastq/sample1.fq.gz, /tmp/RtmpVKt6Bd/filec9f50515d9087/fastq/sample2.fq.gz, /tmp/RtmpVKt6Bd/filec9f50515d9087/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon May 25 00:19:03 2026 ----------
2026-05-25T04:19:03.335114Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:19:03.335593Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50515d9087/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-25T04:19:03.335604Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:19:03.335607Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:19:03.335666Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:19:03.335672Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-25T04:19:03.346609Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:19:03.621662Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:19:03.622132Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50515d9087/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-25T04:19:03.622140Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:19:03.622143Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:19:03.622202Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:19:03.622207Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-25T04:19:03.627440Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:19:03.893992Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:19:03.894333Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50515d9087/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-25T04:19:03.894341Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:19:03.894343Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:19:03.894401Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:19:03.894410Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-25T04:19:03.899339Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:19:04.171331Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:19:04.171692Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50515d9087/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-25T04:19:04.171703Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:19:04.171706Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:19:04.171765Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:19:04.171770Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-25T04:19:04.177554Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f5018a48f69/config_file_827216.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 @ Mon May 25 00:19:04 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f5018a48f69/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/RtmpVKt6Bd/filec9f5018a48f69/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5018a48f69/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5018a48f69/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/RtmpVKt6Bd/filec9f5018a48f69/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/RtmpVKt6Bd/filec9f5018a48f69/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/RtmpVKt6Bd/filec9f5018a48f69/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/RtmpVKt6Bd/filec9f5018a48f69/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/RtmpVKt6Bd/filec9f5018a48f69/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/RtmpVKt6Bd/filec9f5018a48f69/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 @ Mon May 25 00:19:05 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon May 25 00:19:27 2026 ----------------
00:19:27 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_align2genome.bam', and
'/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_align2genome.bam'
parsing /tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.64gene_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, 153610.50Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.70gene_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, 502865.91Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.29gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 559509.10Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.53gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 420658.72Read/s]
-- Running step: isoform_identification @ Mon May 25 00:19:28 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 8 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon May 25 00:19:55 2026 -------------------
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5018a48f69/fastq, /tmp/RtmpVKt6Bd/filec9f5018a48f69/fastq/sample1.fq.gz, /tmp/RtmpVKt6Bd/filec9f5018a48f69/fastq/sample2.fq.gz, /tmp/RtmpVKt6Bd/filec9f5018a48f69/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:20:15 2026 ----------
2026-05-25T04:20:15.395014Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:20:15.395738Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f5018a48f69/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-25T04:20:15.395752Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:20:15.395756Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:20:15.395819Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:20:15.395826Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-25T04:20:15.406681Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:20:15.779710Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:20:15.780247Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-25T04:20:15.780258Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:20:15.780263Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:20:15.780327Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:20:15.780335Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-25T04:20:15.785775Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:20:16.152268Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:20:16.152678Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-25T04:20:16.152692Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:20:16.152696Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:20:16.152759Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:20:16.152766Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-25T04:20:16.158262Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:20:16.490834Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:20:16.491280Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f5018a48f69/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-25T04:20:16.491301Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:20:16.491304Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:20:16.491360Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:20:16.491365Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-25T04:20:16.497000Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f5012d3c740/config_file_827216.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 @ Mon May 25 00:20:17 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5012d3c740/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/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 @ Mon May 25 00:20:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/sample1_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/sample2_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/sample3_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f5012d3c740/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 @ Mon May 25 00:20:21 2026 ----------------
00:20:21 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f5012d3c740/sample1_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f5012d3c740/sample2_align2genome.bam', and
'/tmp/RtmpVKt6Bd/filec9f5012d3c740/sample3_align2genome.bam'
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 164549.62Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.03gene_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, 558674.41Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.43gene_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, 503929.26Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.78gene_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, 427257.76Read/s]
-- Running step: isoform_identification @ Mon May 25 00:20:23 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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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 @ Mon May 25 00:20:46 2026 -------------------
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5012d3c740/fastq, /tmp/RtmpVKt6Bd/filec9f5012d3c740/fastq/sample1.fq.gz, /tmp/RtmpVKt6Bd/filec9f5012d3c740/fastq/sample2.fq.gz, /tmp/RtmpVKt6Bd/filec9f5012d3c740/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample1_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample2_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f5012d3c740/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/RtmpVKt6Bd/filec9f5012d3c740/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f5012d3c740/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 @ Mon May 25 00:20:47 2026 ----------
00:20:47 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f5012d3c740/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample1_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f5012d3c740/sample1_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample2_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f5012d3c740/sample2_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample3_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f5012d3c740/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f5012d3c740/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f5015861eef/config_file_827216.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 @ Mon May 25 00:20:49 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f5015861eef/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/RtmpVKt6Bd/filec9f5015861eef/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5015861eef/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f5015861eef/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/RtmpVKt6Bd/filec9f5015861eef/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/RtmpVKt6Bd/filec9f5015861eef/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/RtmpVKt6Bd/filec9f5015861eef/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/RtmpVKt6Bd/filec9f5015861eef/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/RtmpVKt6Bd/filec9f5015861eef/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/RtmpVKt6Bd/filec9f5015861eef/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 @ Mon May 25 00:20:51 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon May 25 00:21:13 2026 ----------------
00:21:13 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_align2genome.bam', and
'/tmp/RtmpVKt6Bd/filec9f5015861eef/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/RtmpVKt6Bd/filec9f5015861eef/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.18gene_group/s]
/home/biocbuild/bbs-3.24-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, 154723.41Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.42gene_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, 507195.51Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.74gene_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, 492705.57Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.70gene_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, 394750.59Read/s]
-- Running step: isoform_identification @ Mon May 25 00:21:14 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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  |===================================                                   |  50%
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  |====================================================                  |  75%
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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 @ Mon May 25 00:21:38 2026 -------------------
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5015861eef/fastq, /tmp/RtmpVKt6Bd/filec9f5015861eef/fastq/sample1.fq.gz, /tmp/RtmpVKt6Bd/filec9f5015861eef/fastq/sample2.fq.gz, /tmp/RtmpVKt6Bd/filec9f5015861eef/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:21:58 2026 ----------
00:21:58 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f5015861eef/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f5015861eef/sample1_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f5015861eef/sample2_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f5015861eef/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/config_file_827216.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 @ Mon May 25 00:22:00 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/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/RtmpVKt6Bd/filec9f50f4cd9f5/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/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/RtmpVKt6Bd/filec9f50f4cd9f5/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/RtmpVKt6Bd/filec9f50f4cd9f5/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/RtmpVKt6Bd/filec9f50f4cd9f5/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/RtmpVKt6Bd/filec9f50f4cd9f5/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/RtmpVKt6Bd/filec9f50f4cd9f5/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/RtmpVKt6Bd/filec9f50f4cd9f5/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 @ Mon May 25 00:22:02 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample3_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon May 25 00:22:05 2026 ----------------
00:22:05 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_align2genome.bam', and
'/tmp/RtmpVKt6Bd/filec9f50f4cd9f5/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/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.67gene_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, 166029.51Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 565544.47Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.69gene_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, 502167.52Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.92gene_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, 391947.07Read/s]
-- Running step: isoform_identification @ Mon May 25 00:22:06 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:22:07 2026 -------------------
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/fastq, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/fastq/sample1.fq.gz, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/fastq/sample2.fq.gz, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon May 25 00:22:14 2026 ----------
2026-05-25T04:22:14.956816Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:22:14.957989Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-05-25T04:22:14.958007Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:22:14.958026Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:22:14.958156Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:22:14.958168Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-25T04:22:15.000618Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:22:15.549120Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:22:15.549756Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-05-25T04:22:15.549768Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:22:15.549771Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:22:15.549881Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:22:15.549893Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-25T04:22:15.565527Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:22:16.153792Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:22:16.154442Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-05-25T04:22:16.154455Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:22:16.154458Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:22:16.154581Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:22:16.154593Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-25T04:22:16.171109Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:22:16.719192Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:22:16.719766Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f50f4cd9f5/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-05-25T04:22:16.719778Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:22:16.719782Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:22:16.719899Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:22:16.719911Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-25T04:22:16.738934Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f505aef112f/config_file_827216.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 @ Mon May 25 00:22:17 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f505aef112f/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/RtmpVKt6Bd/filec9f505aef112f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f505aef112f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f505aef112f/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/RtmpVKt6Bd/filec9f505aef112f/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/RtmpVKt6Bd/filec9f505aef112f/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/RtmpVKt6Bd/filec9f505aef112f/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/RtmpVKt6Bd/filec9f505aef112f/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/RtmpVKt6Bd/filec9f505aef112f/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/RtmpVKt6Bd/filec9f505aef112f/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 @ Mon May 25 00:22:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon May 25 00:22:41 2026 ----------------
00:22:41 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_align2genome.bam', and
'/tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_align2genome.bam'
parsing /tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.46gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 139110.87Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.84gene_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, 542095.85Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.67gene_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, 490080.39Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 411141.78Read/s]
-- Running step: isoform_identification @ Mon May 25 00:22:42 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:22:43 2026 -------------------
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f505aef112f/fastq, /tmp/RtmpVKt6Bd/filec9f505aef112f/fastq/sample1.fq.gz, /tmp/RtmpVKt6Bd/filec9f505aef112f/fastq/sample2.fq.gz, /tmp/RtmpVKt6Bd/filec9f505aef112f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:23:09 2026 ----------
2026-05-25T04:23:09.698084Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:23:09.698649Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f505aef112f/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-05-25T04:23:09.698663Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:23:09.698667Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:23:09.698789Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:23:09.698803Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-25T04:23:09.740642Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:23:10.447245Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:23:10.447661Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f505aef112f/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-05-25T04:23:10.447673Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:23:10.447676Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:23:10.447799Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:23:10.447812Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-25T04:23:10.463230Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:23:11.064542Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:23:11.065073Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f505aef112f/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-05-25T04:23:11.065083Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:23:11.065087Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:23:11.065206Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:23:11.065219Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-25T04:23:11.081695Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-25T04:23:11.638246Z  INFO oarfish: setting user-provided filter parameters.
2026-05-25T04:23:11.638771Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpVKt6Bd/filec9f505aef112f/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-05-25T04:23:11.638783Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-25T04:23:11.638786Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-25T04:23:11.638906Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-25T04:23:11.638918Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-25T04:23:11.658165Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/config_file_827216.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 @ Mon May 25 00:23:12 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/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 @ Mon May 25 00:23:13 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_matched_reads.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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 @ Mon May 25 00:23:17 2026 ----------------
00:23:17 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_align2genome.bam', and
'/tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_align2genome.bam'
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.67gene_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, 175227.02Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 494424.75Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 505484.00Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.27gene_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, 363609.13Read/s]
-- Running step: isoform_identification @ Mon May 25 00:23:18 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:23:18 2026 -------------------
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/fastq, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/fastq/sample1.fq.gz, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/fastq/sample2.fq.gz, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/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 @ Mon May 25 00:23:22 2026 ----------
00:23:22 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample1_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample2_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f507e9d6cbc/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpVKt6Bd/filec9f50bfd67fb/config_file_827216.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 @ Mon May 25 00:23:25 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpVKt6Bd/filec9f50bfd67fb/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/RtmpVKt6Bd/filec9f50bfd67fb/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f50bfd67fb/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpVKt6Bd/filec9f50bfd67fb/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/RtmpVKt6Bd/filec9f50bfd67fb/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/RtmpVKt6Bd/filec9f50bfd67fb/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/RtmpVKt6Bd/filec9f50bfd67fb/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/RtmpVKt6Bd/filec9f50bfd67fb/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/RtmpVKt6Bd/filec9f50bfd67fb/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/RtmpVKt6Bd/filec9f50bfd67fb/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 @ Mon May 25 00:23:26 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_align2genome.bam
/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_matched_reads.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon May 25 00:23:48 2026 ----------------
00:23:48 Mon May 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_align2genome.bam',
'/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_align2genome.bam', and
'/tmp/RtmpVKt6Bd/filec9f50bfd67fb/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/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 143377.36Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 30.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 536877.78Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.00gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 508277.27Read/s]
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.48gene_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, 357875.77Read/s]
-- Running step: isoform_identification @ Mon May 25 00:23:50 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon May 25 00:23:50 2026 -------------------
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50bfd67fb/fastq, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/fastq/sample1.fq.gz, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/fastq/sample2.fq.gz, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_matched_reads.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_realign2transcript.bam
/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon May 25 00:24:11 2026 ----------
00:24:11 Mon May 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample1_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample2_realign2transcript.bamdone
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_realign2transcript.bam...
parsing /tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpVKt6Bd/filec9f50bfd67fb/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Filtering for genes with at least 2 detected isoforms ...
	6 isoform(s) left.

Aggregating counts by cluster labels ...

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

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  |======================================================================| 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 
814.971  51.818 850.384