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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.4.1.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.2 (2025-10-31)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.4.1’
* 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 ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
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’
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_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
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘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_gene_entropy: no visible global function definition for ‘as’
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 ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
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 ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
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:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file 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 y
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-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     25.098  0.280  25.377
blaze                         4.592 18.456  13.495
find_variants                21.092  0.667  21.156
bulk_long_pipeline            2.366 13.098   2.566
sc_long_multisample_pipeline  8.235  7.088   8.587
sc_plot_genotype             10.941  0.712  10.509
MultiSampleSCPipeline        10.512  0.727  11.637
sc_DTU_analysis               6.914  1.933   6.801
plot_isoform_heatmap          7.292  0.084   7.375
create_sce_from_dir           3.639  2.990   4.105
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 ERROR
Running the tests in ‘tests/testthat.R’ failed.
Last 13 lines of output:
  `expected`: FALSE
  
  experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
  ── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
  Expected `is.null(experiment(result))` to be FALSE.
  Differences:
  `actual`:   TRUE 
  `expected`: FALSE
  
  experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
  
  [ FAIL 12 | WARN 106 | SKIP 0 | PASS 47 ]
  Error:
  ! Test failures.
  Execution halted
* 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: 1 ERROR, 5 NOTEs
See
  ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
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* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.4.1’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R version 4.5.2 (2025-10-31) -- "[Not] Part in a Rumble"
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Platform: x86_64-pc-linux-gnu

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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")
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b554a3381f/config_file_411317.json 
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b554a3381f/config_file_411317.json 
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b554a3381f/config_file_411317.json 
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b532183c07/config_file_411317.json 
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b51f0f28cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b57104512c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b57104512c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b5d423c83/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b5d423c83/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b5d423c83/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b5d423c83/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b5273a4529/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b5151efab1/config_file_411317.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 Dec  8 23:46:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpZxJMiR/file646b5151efab1/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpZxJMiR/file646b5151efab1/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpZxJMiR/file646b5151efab1/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Dec  8 23:46:55 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b57163c292/config_file_411317.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 Dec  8 23:47:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpZxJMiR/file646b57163c292/sample1_align2genome.bam
sample2 ->/tmp/RtmpZxJMiR/file646b57163c292/sample2_align2genome.bam
sample3 ->/tmp/RtmpZxJMiR/file646b57163c292/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Dec  8 23:47:22 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b55b2b82d0/config_file_411317.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 Dec  8 23:47:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpZxJMiR/file646b55b2b82d0/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpZxJMiR/file646b55b2b82d0/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpZxJMiR/file646b55b2b82d0/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Dec  8 23:47:24 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b56e0907d0/config_file_411317.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 Dec  8 23:47:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpZxJMiR/file646b56e0907d0/sample1_align2genome.bam
sample2 ->/tmp/RtmpZxJMiR/file646b56e0907d0/sample2_align2genome.bam
sample3 ->/tmp/RtmpZxJMiR/file646b56e0907d0/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Dec  8 23:47:44 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b5270ecbf2/config_file_411317.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 Dec  8 23:47:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpZxJMiR/file646b5270ecbf2/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpZxJMiR/file646b5270ecbf2/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpZxJMiR/file646b5270ecbf2/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Dec  8 23:47:45 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:47:46 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpZxJMiR/file646b5270ecbf2/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpZxJMiR/file646b5270ecbf2/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpZxJMiR/file646b5270ecbf2/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Dec  8 23:47:47 2025 ----------
2025-12-09T04:47:47.262154Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:47:47.262655Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b5270ecbf2/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-12-09T04:47:47.262669Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:47:47.262673Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:47:47.262747Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:47:47.262756Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-09T04:47:47.265385Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-09T04:47:47.265531Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-12-09T04:47:47.265563Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-12-09T04:47:47.265566Z  INFO oarfish::bulk: number of aligned reads : 98
2025-12-09T04:47:47.265569Z  INFO oarfish::bulk: number of unique alignments : 86
2025-12-09T04:47:47.266256Z  INFO oarfish: oarfish completed successfully.
2025-12-09T04:47:47.274061Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:47:47.274433Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b5270ecbf2/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-12-09T04:47:47.274442Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:47:47.274445Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:47:47.274524Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:47:47.274534Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-09T04:47:47.277234Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-09T04:47:47.277376Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-12-09T04:47:47.277407Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-12-09T04:47:47.277410Z  INFO oarfish::bulk: number of aligned reads : 97
2025-12-09T04:47:47.277413Z  INFO oarfish::bulk: number of unique alignments : 79
2025-12-09T04:47:47.278024Z  INFO oarfish: oarfish completed successfully.
2025-12-09T04:47:47.285783Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:47:47.286283Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b5270ecbf2/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-12-09T04:47:47.286291Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:47:47.286294Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:47:47.286370Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:47:47.286386Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-09T04:47:47.290979Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-09T04:47:47.291155Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-12-09T04:47:47.291190Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-12-09T04:47:47.291192Z  INFO oarfish::bulk: number of aligned reads : 187
2025-12-09T04:47:47.291195Z  INFO oarfish::bulk: number of unique alignments : 140
2025-12-09T04:47:47.291916Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b575affb5/config_file_411317.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 Dec  8 23:47:47 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpZxJMiR/file646b575affb5/sample1_align2genome.bam
sample2 ->/tmp/RtmpZxJMiR/file646b575affb5/sample2_align2genome.bam
sample3 ->/tmp/RtmpZxJMiR/file646b575affb5/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Dec  8 23:48:06 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:48:07 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpZxJMiR/file646b575affb5/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpZxJMiR/file646b575affb5/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpZxJMiR/file646b575affb5/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Dec  8 23:48:27 2025 ----------
2025-12-09T04:48:27.297107Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:48:27.297561Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b575affb5/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-12-09T04:48:27.297573Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:48:27.297577Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:48:27.297663Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:48:27.297674Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-09T04:48:27.300333Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-09T04:48:27.300492Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-12-09T04:48:27.300550Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-12-09T04:48:27.300553Z  INFO oarfish::bulk: number of aligned reads : 98
2025-12-09T04:48:27.300555Z  INFO oarfish::bulk: number of unique alignments : 86
2025-12-09T04:48:27.301216Z  INFO oarfish: oarfish completed successfully.
2025-12-09T04:48:27.313983Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:48:27.314395Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b575affb5/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-12-09T04:48:27.314403Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:48:27.314406Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:48:27.314483Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:48:27.314490Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-09T04:48:27.317154Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-09T04:48:27.317325Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-12-09T04:48:27.317353Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-12-09T04:48:27.317356Z  INFO oarfish::bulk: number of aligned reads : 97
2025-12-09T04:48:27.317358Z  INFO oarfish::bulk: number of unique alignments : 79
2025-12-09T04:48:27.318030Z  INFO oarfish: oarfish completed successfully.
2025-12-09T04:48:27.330226Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:48:27.330782Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b575affb5/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-12-09T04:48:27.330807Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:48:27.330810Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:48:27.330881Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:48:27.330888Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-09T04:48:27.335218Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-12-09T04:48:27.335417Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-12-09T04:48:27.335446Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-12-09T04:48:27.335448Z  INFO oarfish::bulk: number of aligned reads : 187
2025-12-09T04:48:27.335451Z  INFO oarfish::bulk: number of unique alignments : 140
2025-12-09T04:48:27.336169Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b54ed7fce3/config_file_411317.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 Dec  8 23:48:27 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpZxJMiR/file646b54ed7fce3/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpZxJMiR/file646b54ed7fce3/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpZxJMiR/file646b54ed7fce3/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Dec  8 23:48:28 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:48:28 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpZxJMiR/file646b54ed7fce3/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpZxJMiR/file646b54ed7fce3/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpZxJMiR/file646b54ed7fce3/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Dec  8 23:48:29 2025 ----------
23:48:29 Mon Dec 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b55b7ebc53/config_file_411317.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 Dec  8 23:48:30 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpZxJMiR/file646b55b7ebc53/sample1_align2genome.bam
sample2 ->/tmp/RtmpZxJMiR/file646b55b7ebc53/sample2_align2genome.bam
sample3 ->/tmp/RtmpZxJMiR/file646b55b7ebc53/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Dec  8 23:48:49 2025 -------------
Inputs:  ['/tmp/RtmpZxJMiR/file646b54ed7fce3/sample1_realign2transcript.bam', '/tmp/RtmpZxJMiR/file646b54ed7fce3/sample2_realign2transcript.bam', '/tmp/RtmpZxJMiR/file646b54ed7fce3/sample3_realign2transcript.bam'] /tmp/RtmpZxJMiR/file646b54ed7fce3/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:48:50 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpZxJMiR/file646b55b7ebc53/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpZxJMiR/file646b55b7ebc53/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpZxJMiR/file646b55b7ebc53/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Dec  8 23:49:09 2025 ----------
23:49:09 Mon Dec 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpZxJMiR/file646b55b7ebc53/sample1_realign2transcript.bam', '/tmp/RtmpZxJMiR/file646b55b7ebc53/sample2_realign2transcript.bam', '/tmp/RtmpZxJMiR/file646b55b7ebc53/sample3_realign2transcript.bam'] /tmp/RtmpZxJMiR/file646b55b7ebc53/transcript_assembly.fa.fai 5 0.4 0.4
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b571d7ef21/config_file_411317.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Dec  8 23:49:10 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b571d7ef21/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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 Dec  8 23:49:10 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpZxJMiR/file646b571d7ef21/matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b571d7ef21/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Dec  8 23:49:11 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b57430795a/config_file_411317.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Dec  8 23:49:11 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b57430795a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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 Dec  8 23:49:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b57430795a/matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b57430795a/align2genome.bam
-- Running step: isoform_identification @ Mon Dec  8 23:49:31 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b56b093067/config_file_411317.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Dec  8 23:49:31 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b56b093067/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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 Dec  8 23:49:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpZxJMiR/file646b56b093067/matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b56b093067/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: isoform_identification @ Mon Dec  8 23:49:32 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b57b48c19c/config_file_411317.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Dec  8 23:49:32 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b57b48c19c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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 Dec  8 23:49:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b57b48c19c/matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b57b48c19c/align2genome.bam
-- Running step: isoform_identification @ Mon Dec  8 23:49:51 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b53d99066f/config_file_411317.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Dec  8 23:49:51 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b53d99066f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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 Dec  8 23:49:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpZxJMiR/file646b53d99066f/matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b53d99066f/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Dec  8 23:49:52 2025 -------------
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:49:52 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b53d99066f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b53d99066f/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpZxJMiR/file646b53d99066f/matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b53d99066f/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 Dec  8 23:49:53 2025 ----------
2025-12-09T04:49:53.126044Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:49:53.126517Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b53d99066f/realign2transcript.bam, contains 10 reference sequences.
2025-12-09T04:49:53.126529Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:49:53.126544Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:49:53.126617Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:49:53.126624Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-09T04:49:53.137269Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b550009030/config_file_411317.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Dec  8 23:49:54 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b550009030/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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 Dec  8 23:49:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b550009030/matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b550009030/align2genome.bam
-- Running step: isoform_identification @ Mon Dec  8 23:50:11 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:50:12 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b550009030/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b550009030/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b550009030/matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b550009030/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Dec  8 23:50:31 2025 ----------
2025-12-09T04:50:31.360518Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:50:31.361066Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b550009030/realign2transcript.bam, contains 10 reference sequences.
2025-12-09T04:50:31.361077Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:50:31.361081Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:50:31.361154Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:50:31.361163Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-12-09T04:50:31.372400Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b51670cd9a/config_file_411317.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Dec  8 23:50:32 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b51670cd9a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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 Dec  8 23:50:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpZxJMiR/file646b51670cd9a/matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b51670cd9a/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: isoform_identification @ Mon Dec  8 23:50:32 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:50:33 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b51670cd9a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b51670cd9a/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpZxJMiR/file646b51670cd9a/matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b51670cd9a/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 Dec  8 23:50:33 2025 ----------
23:50:33 Mon Dec 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b54488e626/config_file_411317.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Dec  8 23:50:34 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b54488e626/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
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 Dec  8 23:50:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b54488e626/matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b54488e626/align2genome.bam
-- Running step: isoform_identification @ Mon Dec  8 23:50:52 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:50:53 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b54488e626/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b54488e626/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b54488e626/matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b54488e626/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Dec  8 23:51:12 2025 ----------
23:51:12 Mon Dec 08 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b54f8c2447/config_file_411317.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 Dec  8 23:51:13 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b54f8c2447/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b54f8c2447/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b54f8c2447/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b54f8c2447/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b54f8c2447/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b54f8c2447/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b54f8c2447/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b54f8c2447/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b54f8c2447/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b54f8c2447/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Dec  8 23:51:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpZxJMiR/file646b54f8c2447/sampleA_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b54f8c2447/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpZxJMiR/file646b54f8c2447/sample1_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b54f8c2447/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpZxJMiR/file646b54f8c2447/sample2_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b54f8c2447/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpZxJMiR/file646b54f8c2447/sample3_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b54f8c2447/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Dec  8 23:51:16 2025 ----------------
23:51:16 Mon Dec 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpZxJMiR/file646b54f8c2447/sampleA_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b54f8c2447/sample1_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b54f8c2447/sample2_align2genome.bam', and
'/tmp/RtmpZxJMiR/file646b54f8c2447/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpZxJMiR/file646b54f8c2447/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.11gene_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, 418159.20Read/s]
parsing /tmp/RtmpZxJMiR/file646b54f8c2447/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.17gene_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, 1290396.26Read/s]
parsing /tmp/RtmpZxJMiR/file646b54f8c2447/sample2_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1217716.87Read/s]
parsing /tmp/RtmpZxJMiR/file646b54f8c2447/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.40gene_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, 725106.15Read/s]
-- Running step: isoform_identification @ Mon Dec  8 23:51:17 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b579972663/config_file_411317.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 Dec  8 23:51:18 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b579972663/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b579972663/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b579972663/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b579972663/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b579972663/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b579972663/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b579972663/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b579972663/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b579972663/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b579972663/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Dec  8 23:51:19 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b579972663/sampleA_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b579972663/sampleA_align2genome.bam
/tmp/RtmpZxJMiR/file646b579972663/sample1_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b579972663/sample1_align2genome.bam
/tmp/RtmpZxJMiR/file646b579972663/sample2_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b579972663/sample2_align2genome.bam
/tmp/RtmpZxJMiR/file646b579972663/sample3_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b579972663/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Dec  8 23:51:38 2025 ----------------
23:51:38 Mon Dec 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpZxJMiR/file646b579972663/sampleA_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b579972663/sample1_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b579972663/sample2_align2genome.bam', and
'/tmp/RtmpZxJMiR/file646b579972663/sample3_align2genome.bam'
parsing /tmp/RtmpZxJMiR/file646b579972663/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.79gene_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, 427327.41Read/s]
parsing /tmp/RtmpZxJMiR/file646b579972663/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1425470.36Read/s]
parsing /tmp/RtmpZxJMiR/file646b579972663/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.25gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1372661.34Read/s]
parsing /tmp/RtmpZxJMiR/file646b579972663/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.29gene_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, 776607.91Read/s]
-- Running step: isoform_identification @ Mon Dec  8 23:51:39 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b57837e001/config_file_411317.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 Dec  8 23:51:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b57837e001/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b57837e001/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b57837e001/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b57837e001/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b57837e001/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b57837e001/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b57837e001/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b57837e001/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b57837e001/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b57837e001/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Dec  8 23:51:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpZxJMiR/file646b57837e001/sampleA_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b57837e001/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/RtmpZxJMiR/file646b57837e001/sample1_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b57837e001/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/RtmpZxJMiR/file646b57837e001/sample2_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b57837e001/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/RtmpZxJMiR/file646b57837e001/sample3_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b57837e001/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 Dec  8 23:51:42 2025 ----------------
23:51:42 Mon Dec 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpZxJMiR/file646b57837e001/sampleA_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b57837e001/sample1_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b57837e001/sample2_align2genome.bam', and
'/tmp/RtmpZxJMiR/file646b57837e001/sample3_align2genome.bam'
parsing /tmp/RtmpZxJMiR/file646b57837e001/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.41gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 406850.58Read/s]
parsing /tmp/RtmpZxJMiR/file646b57837e001/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.97gene_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, 1291349.75Read/s]
parsing /tmp/RtmpZxJMiR/file646b57837e001/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.75gene_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, 1306962.48Read/s]
parsing /tmp/RtmpZxJMiR/file646b57837e001/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.98gene_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, 760278.42Read/s]
-- Running step: isoform_identification @ Mon Dec  8 23:51:43 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b524125546/config_file_411317.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 Dec  8 23:51:43 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b524125546/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b524125546/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b524125546/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b524125546/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b524125546/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b524125546/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b524125546/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b524125546/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b524125546/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b524125546/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Dec  8 23:51:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b524125546/sampleA_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b524125546/sampleA_align2genome.bam
/tmp/RtmpZxJMiR/file646b524125546/sample1_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b524125546/sample1_align2genome.bam
/tmp/RtmpZxJMiR/file646b524125546/sample2_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b524125546/sample2_align2genome.bam
/tmp/RtmpZxJMiR/file646b524125546/sample3_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b524125546/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Dec  8 23:52:04 2025 ----------------
23:52:04 Mon Dec 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpZxJMiR/file646b524125546/sampleA_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b524125546/sample1_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b524125546/sample2_align2genome.bam', and
'/tmp/RtmpZxJMiR/file646b524125546/sample3_align2genome.bam'
parsing /tmp/RtmpZxJMiR/file646b524125546/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.99gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 427833.04Read/s]
parsing /tmp/RtmpZxJMiR/file646b524125546/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  9.21gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  9.19gene_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, 1279687.58Read/s]
parsing /tmp/RtmpZxJMiR/file646b524125546/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.93gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1340547.17Read/s]
parsing /tmp/RtmpZxJMiR/file646b524125546/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.71gene_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, 760830.07Read/s]
-- Running step: isoform_identification @ Mon Dec  8 23:52:05 2025 -------------
Unzipping annotation file for bambu
Saving _problems/test-run_FLAMES-32.R
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b545c1bca9/config_file_411317.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 Dec  8 23:52:05 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b545c1bca9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b545c1bca9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b545c1bca9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b545c1bca9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Dec  8 23:52:06 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpZxJMiR/file646b545c1bca9/sample1_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b545c1bca9/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpZxJMiR/file646b545c1bca9/sample2_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b545c1bca9/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpZxJMiR/file646b545c1bca9/sample3_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b545c1bca9/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Dec  8 23:52:07 2025 ----------------
23:52:07 Mon Dec 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b545c1bca9/sample1_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b545c1bca9/sample2_align2genome.bam', and
'/tmp/RtmpZxJMiR/file646b545c1bca9/sample3_align2genome.bam'
parsing /tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.66gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 432473.81Read/s]
parsing /tmp/RtmpZxJMiR/file646b545c1bca9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.03gene_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, 1390315.57Read/s]
parsing /tmp/RtmpZxJMiR/file646b545c1bca9/sample2_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1283132.65Read/s]
parsing /tmp/RtmpZxJMiR/file646b545c1bca9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 723255.62Read/s]
-- Running step: isoform_identification @ Mon Dec  8 23:52:08 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:52:09 2025 -------------------
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b545c1bca9/fastq, /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample1.fq.gz, /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample2.fq.gz, /tmp/RtmpZxJMiR/file646b545c1bca9/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b545c1bca9/sample1_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b545c1bca9/sample2_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b545c1bca9/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b545c1bca9/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b545c1bca9/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b545c1bca9/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpZxJMiR/file646b545c1bca9/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpZxJMiR/file646b545c1bca9/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpZxJMiR/file646b545c1bca9/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpZxJMiR/file646b545c1bca9/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpZxJMiR/file646b545c1bca9/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpZxJMiR/file646b545c1bca9/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Dec  8 23:52:11 2025 ----------
2025-12-09T04:52:11.188217Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:52:11.188622Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b545c1bca9/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-12-09T04:52:11.188634Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:52:11.188637Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:52:11.188720Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:52:11.188728Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-09T04:52:11.200893Z  INFO oarfish::single_cell: Processed 100 cells.
2025-12-09T04:52:11.883992Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:52:11.884363Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b545c1bca9/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-12-09T04:52:11.884369Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:52:11.884372Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:52:11.884448Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:52:11.884455Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-09T04:52:12.471822Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:52:12.472195Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b545c1bca9/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-12-09T04:52:12.472203Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:52:12.472206Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:52:12.472288Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:52:12.472296Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-09T04:52:13.115475Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:52:13.115936Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b545c1bca9/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-12-09T04:52:13.115946Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:52:13.115949Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:52:13.116027Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:52:13.116034Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b567cd66c8/config_file_411317.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 Dec  8 23:52:13 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b567cd66c8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b567cd66c8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b567cd66c8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b567cd66c8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Dec  8 23:52:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_align2genome.bam
/tmp/RtmpZxJMiR/file646b567cd66c8/sample1_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b567cd66c8/sample1_align2genome.bam
/tmp/RtmpZxJMiR/file646b567cd66c8/sample2_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b567cd66c8/sample2_align2genome.bam
/tmp/RtmpZxJMiR/file646b567cd66c8/sample3_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b567cd66c8/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Dec  8 23:52:33 2025 ----------------
23:52:33 Mon Dec 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b567cd66c8/sample1_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b567cd66c8/sample2_align2genome.bam', and
'/tmp/RtmpZxJMiR/file646b567cd66c8/sample3_align2genome.bam'
parsing /tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.42gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:705: 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, 446944.29Read/s]
parsing /tmp/RtmpZxJMiR/file646b567cd66c8/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.17gene_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, 1468799.55Read/s]
parsing /tmp/RtmpZxJMiR/file646b567cd66c8/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 40.39gene_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, 1365333.33Read/s]
parsing /tmp/RtmpZxJMiR/file646b567cd66c8/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.92gene_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, 788758.84Read/s]
-- Running step: isoform_identification @ Mon Dec  8 23:52:34 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:52:35 2025 -------------------
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b567cd66c8/fastq, /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample1.fq.gz, /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample2.fq.gz, /tmp/RtmpZxJMiR/file646b567cd66c8/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b567cd66c8/sample1_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b567cd66c8/sample2_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b567cd66c8/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b567cd66c8/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b567cd66c8/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b567cd66c8/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_realign2transcript.bam
/tmp/RtmpZxJMiR/file646b567cd66c8/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpZxJMiR/file646b567cd66c8/sample1_realign2transcript.bam
/tmp/RtmpZxJMiR/file646b567cd66c8/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpZxJMiR/file646b567cd66c8/sample2_realign2transcript.bam
/tmp/RtmpZxJMiR/file646b567cd66c8/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpZxJMiR/file646b567cd66c8/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Dec  8 23:52:55 2025 ----------
2025-12-09T04:52:55.886361Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:52:55.886864Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b567cd66c8/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-12-09T04:52:55.886876Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:52:55.886880Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:52:55.886974Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:52:55.886982Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-09T04:52:55.898556Z  INFO oarfish::single_cell: Processed 100 cells.
2025-12-09T04:52:56.584643Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:52:56.585016Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b567cd66c8/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-12-09T04:52:56.585023Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:52:56.585027Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:52:56.585109Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:52:56.585117Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-09T04:52:57.256730Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:52:57.257081Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b567cd66c8/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-12-09T04:52:57.257089Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:52:57.257092Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:52:57.257174Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:52:57.257181Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-12-09T04:52:57.817966Z  INFO oarfish: setting user-provided filter parameters.
2025-12-09T04:52:57.818362Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpZxJMiR/file646b567cd66c8/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-12-09T04:52:57.818374Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-12-09T04:52:57.818377Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-12-09T04:52:57.818460Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-12-09T04:52:57.818468Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b543beec21/config_file_411317.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 Dec  8 23:52:58 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b543beec21/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b543beec21/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b543beec21/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b543beec21/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Dec  8 23:52:59 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpZxJMiR/file646b543beec21/sampleA_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b543beec21/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/RtmpZxJMiR/file646b543beec21/sample1_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b543beec21/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/RtmpZxJMiR/file646b543beec21/sample2_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b543beec21/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/RtmpZxJMiR/file646b543beec21/sample3_matched_reads.fastq.gz -> /tmp/RtmpZxJMiR/file646b543beec21/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 Dec  8 23:53:00 2025 ----------------
23:53:00 Mon Dec 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpZxJMiR/file646b543beec21/sampleA_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b543beec21/sample1_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b543beec21/sample2_align2genome.bam', and
'/tmp/RtmpZxJMiR/file646b543beec21/sample3_align2genome.bam'
parsing /tmp/RtmpZxJMiR/file646b543beec21/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.01gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 426805.60Read/s]
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.50gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1397542.32Read/s]
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.90gene_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, 1163145.87Read/s]
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.72gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 766278.87Read/s]
-- Running step: isoform_identification @ Mon Dec  8 23:53:01 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:53:02 2025 -------------------
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b543beec21/fastq, /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample1.fq.gz, /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample2.fq.gz, /tmp/RtmpZxJMiR/file646b543beec21/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b543beec21/sampleA_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b543beec21/sample1_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b543beec21/sample2_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b543beec21/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b543beec21/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b543beec21/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b543beec21/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b543beec21/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpZxJMiR/file646b543beec21/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpZxJMiR/file646b543beec21/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/RtmpZxJMiR/file646b543beec21/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpZxJMiR/file646b543beec21/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/RtmpZxJMiR/file646b543beec21/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpZxJMiR/file646b543beec21/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/RtmpZxJMiR/file646b543beec21/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpZxJMiR/file646b543beec21/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 Dec  8 23:53:03 2025 ----------
23:53:03 Mon Dec 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpZxJMiR/file646b543beec21/sampleA_realign2transcript.bam...
parsing /tmp/RtmpZxJMiR/file646b543beec21/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpZxJMiR/file646b543beec21/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample1_realign2transcript.bam...
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpZxJMiR/file646b543beec21/sample1_realign2transcript.bamdone
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample2_realign2transcript.bam...
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpZxJMiR/file646b543beec21/sample2_realign2transcript.bamdone
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample3_realign2transcript.bam...
parsing /tmp/RtmpZxJMiR/file646b543beec21/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpZxJMiR/file646b543beec21/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpZxJMiR/file646b5bac3a59/config_file_411317.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 Dec  8 23:53:06 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b5bac3a59/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b5bac3a59/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b5bac3a59/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpZxJMiR/file646b5bac3a59/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Dec  8 23:53:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_align2genome.bam
/tmp/RtmpZxJMiR/file646b5bac3a59/sample1_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b5bac3a59/sample1_align2genome.bam
/tmp/RtmpZxJMiR/file646b5bac3a59/sample2_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b5bac3a59/sample2_align2genome.bam
/tmp/RtmpZxJMiR/file646b5bac3a59/sample3_matched_reads.fastq.gz ->/tmp/RtmpZxJMiR/file646b5bac3a59/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Dec  8 23:53:27 2025 ----------------
23:53:27 Mon Dec 08 2025 quantify genes 
Using BAM(s): '/tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b5bac3a59/sample1_align2genome.bam',
'/tmp/RtmpZxJMiR/file646b5bac3a59/sample2_align2genome.bam', and
'/tmp/RtmpZxJMiR/file646b5bac3a59/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.19gene_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, 409088.64Read/s]
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample1_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1271926.25Read/s]
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.78gene_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, 1369971.26Read/s]
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.98gene_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, 777990.80Read/s]
-- Running step: isoform_identification @ Mon Dec  8 23:53:27 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Dec  8 23:53:28 2025 -------------------
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b5bac3a59/fastq, /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample1.fq.gz, /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample2.fq.gz, /tmp/RtmpZxJMiR/file646b5bac3a59/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b5bac3a59/sample1_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b5bac3a59/sample2_matched_reads.fastq.gz, /tmp/RtmpZxJMiR/file646b5bac3a59/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b5bac3a59/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b5bac3a59/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpZxJMiR/file646b5bac3a59/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_realign2transcript.bam
/tmp/RtmpZxJMiR/file646b5bac3a59/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpZxJMiR/file646b5bac3a59/sample1_realign2transcript.bam
/tmp/RtmpZxJMiR/file646b5bac3a59/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpZxJMiR/file646b5bac3a59/sample2_realign2transcript.bam
/tmp/RtmpZxJMiR/file646b5bac3a59/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpZxJMiR/file646b5bac3a59/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Dec  8 23:53:47 2025 ----------
23:53:47 Mon Dec 08 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_realign2transcript.bam...
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpZxJMiR/file646b5bac3a59/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample1_realign2transcript.bam...
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpZxJMiR/file646b5bac3a59/sample1_realign2transcript.bamdone
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample2_realign2transcript.bam...
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpZxJMiR/file646b5bac3a59/sample2_realign2transcript.bamdone
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample3_realign2transcript.bam...
parsing /tmp/RtmpZxJMiR/file646b5bac3a59/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpZxJMiR/file646b5bac3a59/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 12 | WARN 106 | SKIP 0 | PASS 47 ]

══ Failed tests ════════════════════════════════════════════════════════════════
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
── Failure ('test-run_FLAMES.R:29:11'): run_FLAMES() completes and returns experiment for all pipeline types and option combinations ──
Expected `is.null(experiment(result))` to be FALSE.
Differences:
`actual`:   TRUE 
`expected`: FALSE

experiment is NULL for run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)

[ FAIL 12 | WARN 106 | SKIP 0 | PASS 47 ]
Error:
! Test failures.
Execution halted