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###
### Running command:
###
###   /home/biocbuild/bbs-3.24-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.24-bioc/R/site-library --timings FLAMES_2.7.0.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.24-bioc/meat/FLAMES.Rcheck’
* using R version 4.6.0 RC (2026-04-17 r89917)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0
* running under: Ubuntu 24.04.4 LTS
* using session charset: UTF-8
* current time: 2026-05-09 03:49:46 UTC
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.7.0’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... OK
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.24-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
* checking C++ specification ... INFO
  specified C++17
* checking installed package size ... INFO
  installed size is 13.3Mb
  sub-directories of 1Mb or more:
    bin    8.3Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
Namespace in Imports field not imported from: ‘scuttle’
  All declared Imports should be used.
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  ‘gene_quantification’ ‘isoform_identification’ ‘minimap2_align’
  ‘transcript_quantification’
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
barcode_group: no visible global function definition for ‘new’
barcode_segment: no visible global function definition for ‘new’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_diversity: no visible global function definition for ‘as’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable ‘CB’
plot_demultiplex_raw: no visible binding for global variable ‘UB’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_isoforms: no visible binding for global variable ‘xmin’
plot_isoforms: no visible binding for global variable ‘xmax’
plot_isoforms: no visible binding for global variable ‘ymin’
plot_isoforms: no visible binding for global variable ‘ymax’
plot_isoforms: no visible binding for global variable ‘x’
plot_isoforms: no visible binding for global variable ‘xend’
plot_isoforms: no visible binding for global variable ‘y’
plot_isoforms: no visible binding for global variable ‘yend’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_files’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  CB Count FSM_match FlankEditDist Freq Reads Sample Type UB UMI_count
  adj.p.value allele allele_count as bam_index barcode barcode_rank
  barcodes_file barcodes_files capture.output cell_total_reads
  chisq.test count counts_no_ins cumpct demultiplexed reads
  demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x xend xmax xmin y yend ymax ymin
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
find_variants                22.006  1.139  22.085
plot_isoform_reduced_dim     20.861  0.816  21.675
blaze                         4.850 15.239  13.013
bulk_long_pipeline            2.386 13.432   2.468
sc_long_multisample_pipeline  8.323  4.821   7.731
sc_plot_genotype             11.396  1.666  11.166
MultiSampleSCPipeline        10.084  0.715  10.892
create_sce_from_dir           5.734  2.898   6.481
sc_DTU_analysis               6.769  1.622   6.519
create_se_from_dir            5.208  0.149   5.345
plot_durations                5.104  0.100   5.192
run_FLAMES                    4.923  0.101   5.010
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

Status: 4 NOTEs
See
  ‘/home/biocbuild/bbs-3.24-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.24-bioc/R/bin/R CMD INSTALL FLAMES
###
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* installing to library ‘/home/biocbuild/bbs-3.24-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.7.0’
** using non-staged installation via StagedInstall field
** libs
specified C++17
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:142:56: warning: comparison of integer expressions of different signedness: ‘__gnu_cxx::__alloc_traits<std::allocator<unsigned int>, unsigned int>::value_type’ {aka ‘unsigned int’} and ‘int’ [-Wsign-compare]
  142 |       if (j == (len2 - 1) && dist_holder[i * len2 + j] < best) {
main-functions/flexiplex.cpp: In function ‘void refine_matched_segments(const std::string&, const std::vector<Segment>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_set<std::__cxx11::basic_string<char> > >*, const std::vector<int>&, Barcode&, std::vector<int>&, std::vector<int>&)’:
main-functions/flexiplex.cpp:348:70: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘const int’ [-Wsign-compare]
  348 |         } else if (editDistance < best_edit_distance && editDistance <= s.max_edit_distance) {
      |                                                         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:356:30: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘const int’ [-Wsign-compare]
  356 |       if (best_edit_distance <= s.max_edit_distance && current_segment_unambiguous) {
      |           ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, bool, bool, bool, const std::vector<Segment>&, const std::unordered_map<std::__cxx11::basic_string<char>, BarcodeGroup>&)’:
main-functions/flexiplex.cpp:790:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const size_t’ {aka ‘const long unsigned int’} [-Wsign-compare]
  790 |   for (int b = 0; b < vec_size; b++) {
      |                   ~~^~~~~~~~~~
main-functions/flexiplex.cpp:803:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const size_t’ {aka ‘const long unsigned int’} [-Wsign-compare]
  803 |     for (int k = 0; k < vec_size; k++) {
      |                     ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.24-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.24-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.24-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.24-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.24-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Building oarfish with cargo
mkdir -p ../inst/bin
mkdir cargo_temp
(cargo install oarfish --root cargo_temp --bin oarfish --vers 0.8.0)
    Updating crates.io index
  Installing oarfish v0.8.0
    Updating crates.io index
     Locking 279 packages to latest compatible versions
      Adding generic-array v0.14.7 (available: v0.14.9)
      Adding indicatif v0.17.11 (available: v0.18.4)
      Adding needletail v0.6.3 (available: v0.7.3)
      Adding noodles-bam v0.78.0 (available: v0.89.0)
      Adding noodles-bgzf v0.38.0 (available: v0.47.0)
      Adding noodles-sam v0.74.0 (available: v0.85.0)
      Adding rand v0.9.4 (available: v0.10.1)
      Adding tabled v0.18.0 (available: v0.20.0)
      Adding typed-builder v0.21.2 (available: v0.23.2)
 Downloading crates ...
  Downloaded cc v1.2.62
   Compiling libc v0.2.186
   Compiling proc-macro2 v1.0.106
   Compiling quote v1.0.45
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   Compiling flate2 v1.1.9
   Compiling ndarray v0.16.1
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   Compiling alga v0.9.3
   Compiling zstd-sys v2.0.16+zstd.1.5.7
   Compiling bzip2-sys v0.1.13+1.0.8
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   Compiling lz4-sys v1.11.1+lz4-1.10.0
   Compiling minimap2-sys v0.1.30+minimap2.2.30
   Compiling noodles-bgzf v0.38.0
   Compiling sha2 v0.10.9
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   Compiling proc-macro-error2 v2.0.1
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   Compiling ppv-lite86 v0.2.21
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   Compiling serde_derive v1.0.228
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   Compiling async-stream-impl v0.3.6
   Compiling tracing-attributes v0.1.31
   Compiling async-trait v0.1.89
   Compiling thiserror-impl v1.0.69
   Compiling tabled_derive v0.10.0
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   Compiling rand_chacha v0.3.1
   Compiling rand_chacha v0.9.0
   Compiling async-stream v0.3.6
   Compiling rand v0.9.4
   Compiling rand v0.8.6
   Compiling futures-util v0.3.32
   Compiling tabled v0.18.0
   Compiling bzip2 v0.4.4
   Compiling bytemuck v1.25.0
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   Compiling safe_arch v0.7.4
   Compiling bstr v1.12.1
   Compiling matchers v0.2.0
   Compiling regex v1.12.3
   Compiling tracing-subscriber v0.3.23
   Compiling rand_distr v0.4.3
   Compiling wide v0.7.33
   Compiling clap v4.6.1
   Compiling noodles-core v0.17.0
   Compiling noodles-csi v0.46.0
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   Compiling bincode v1.3.3
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   Compiling minimap2 v0.1.31+minimap2.2.30
   Compiling statrs v0.18.0
   Compiling oarfish v0.8.0
    Finished `release` profile [optimized] target(s) in 47.69s
  Installing /home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/cargo_temp/bin/oarfish
   Installed package `oarfish v0.8.0` (executable `oarfish`)
warning: be sure to add `/home/biocbuild/bbs-3.24-bioc/meat/FLAMES/src/cargo_temp/bin` to your PATH to be able to run the installed binaries
cp cargo_temp/bin/oarfish ../inst/bin/
cargo uninstall oarfish --root cargo_temp
    Removing cargo_temp/bin/oarfish
rm -rf cargo_temp
installing to /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

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

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

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
i Writing configuration to: /tmp/RtmpUC1bK6/file27797826ea8270/config_file_2587000.json
i Writing configuration to: /tmp/RtmpUC1bK6/file27797826ea8270/config_file_2587000.json
i Writing configuration to: /tmp/RtmpUC1bK6/file27797826ea8270/config_file_2587000.json
i Writing configuration to: /tmp/RtmpUC1bK6/file27797868d3190/config_file_2587000.json
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpUC1bK6/file277978650d98a4/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpUC1bK6/file277978367370e7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpUC1bK6/file277978367370e7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779785017228c/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779785017228c/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779785017228c/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779785017228c/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Converting legacy `pattern` argument to `segments`...
Loading known barcodes from /tmp/RtmpUC1bK6/file27797872db40dc/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
2 transcripts found in the BAM file.
1(50%) transcripts failed the filter.
Failed transcripts account for 100 reads, out of 200(50%) reads in total.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779786f1522f9/config_file_2587000.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri May  8 23:58:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpUC1bK6/file2779786f1522f9/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpUC1bK6/file2779786f1522f9/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpUC1bK6/file2779786f1522f9/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri May  8 23:59:00 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri May  8 23:59:24 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpUC1bK6/file2779786f1522f9/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpUC1bK6/file2779786f1522f9/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpUC1bK6/file2779786f1522f9/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri May  8 23:59:25 2026 ----------
2026-05-09T03:59:25.857531Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T03:59:25.857909Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779786f1522f9/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-09T03:59:25.857921Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T03:59:25.857925Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T03:59:25.857991Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T03:59:25.857998Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-09T03:59:25.862143Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-09T03:59:25.862357Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 686   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 4     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-05-09T03:59:25.862392Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-09T03:59:25.862395Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-09T03:59:25.862397Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-09T03:59:25.863233Z  INFO oarfish: oarfish completed successfully.
2026-05-09T03:59:25.870319Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T03:59:25.870818Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779786f1522f9/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-09T03:59:25.870832Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T03:59:25.870835Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T03:59:25.870890Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T03:59:25.870895Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-09T03:59:25.875007Z  INFO oarfish::alignment_parser: the alignment file contained 1 unmapped read records.
2026-05-09T03:59:25.875204Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 695   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-05-09T03:59:25.875225Z  INFO oarfish::bulk: Total number of alignment records : 369
2026-05-09T03:59:25.875228Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-09T03:59:25.875230Z  INFO oarfish::bulk: number of unique alignments : 235
2026-05-09T03:59:25.876063Z  INFO oarfish: oarfish completed successfully.
2026-05-09T03:59:25.882986Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T03:59:25.883433Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779786f1522f9/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-09T03:59:25.883445Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T03:59:25.883448Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T03:59:25.883502Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T03:59:25.883506Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-09T03:59:25.887564Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-09T03:59:25.887762Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 696   │
│ aligned fraction too low        │ 14    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 284   │
╰─────────────────────────────────┴───────╯

2026-05-09T03:59:25.887790Z  INFO oarfish::bulk: Total number of alignment records : 362
2026-05-09T03:59:25.887793Z  INFO oarfish::bulk: number of aligned reads : 284
2026-05-09T03:59:25.887796Z  INFO oarfish::bulk: number of unique alignments : 237
2026-05-09T03:59:25.888656Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797879364918/config_file_2587000.json
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Fri May  8 23:59:26 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpUC1bK6/file27797879364918/sample1_align2genome.bam
sample2 ->/tmp/RtmpUC1bK6/file27797879364918/sample2_align2genome.bam
sample3 ->/tmp/RtmpUC1bK6/file27797879364918/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri May  8 23:59:48 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:00:10 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpUC1bK6/file27797879364918/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpUC1bK6/file27797879364918/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpUC1bK6/file27797879364918/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:00:32 2026 ----------
2026-05-09T04:00:32.580261Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:00:32.580740Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797879364918/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-05-09T04:00:32.580758Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:00:32.580763Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:00:32.580838Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:00:32.580846Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-09T04:00:32.584966Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-09T04:00:32.585238Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 702   │
│ aligned fraction too low        │ 13    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 285   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:00:32.585268Z  INFO oarfish::bulk: Total number of alignment records : 364
2026-05-09T04:00:32.585272Z  INFO oarfish::bulk: number of aligned reads : 285
2026-05-09T04:00:32.585276Z  INFO oarfish::bulk: number of unique alignments : 238
2026-05-09T04:00:32.586194Z  INFO oarfish: oarfish completed successfully.
2026-05-09T04:00:32.597383Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:00:32.597811Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797879364918/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-05-09T04:00:32.597826Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:00:32.597831Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:00:32.597909Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:00:32.597916Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-09T04:00:32.601842Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-09T04:00:32.602118Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 687   │
│ aligned fraction too low        │ 16    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 6     │
│ reads with valid best alignment │ 282   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:00:32.602145Z  INFO oarfish::bulk: Total number of alignment records : 355
2026-05-09T04:00:32.602148Z  INFO oarfish::bulk: number of aligned reads : 282
2026-05-09T04:00:32.602150Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-09T04:00:32.603022Z  INFO oarfish: oarfish completed successfully.
2026-05-09T04:00:32.614071Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:00:32.614595Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797879364918/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-05-09T04:00:32.614609Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:00:32.614612Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:00:32.614677Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:00:32.614684Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-09T04:00:32.618890Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-05-09T04:00:32.619144Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 688   │
│ aligned fraction too low        │ 15    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 7     │
│ reads with valid best alignment │ 283   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:00:32.619170Z  INFO oarfish::bulk: Total number of alignment records : 354
2026-05-09T04:00:32.619173Z  INFO oarfish::bulk: number of aligned reads : 283
2026-05-09T04:00:32.619175Z  INFO oarfish::bulk: number of unique alignments : 240
2026-05-09T04:00:32.620055Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797817346022/config_file_2587000.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 @ Sat May  9 00:00:32 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpUC1bK6/file27797817346022/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpUC1bK6/file27797817346022/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpUC1bK6/file27797817346022/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat May  9 00:00:34 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:00:55 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpUC1bK6/file27797817346022/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpUC1bK6/file27797817346022/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpUC1bK6/file27797817346022/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat May  9 00:00:56 2026 ----------
00:00:56 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797851fa0347/config_file_2587000.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 @ Sat May  9 00:00:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpUC1bK6/file27797851fa0347/sample1_align2genome.bam
sample2 ->/tmp/RtmpUC1bK6/file27797851fa0347/sample2_align2genome.bam
sample3 ->/tmp/RtmpUC1bK6/file27797851fa0347/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat May  9 00:01:20 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 5 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:01:41 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpUC1bK6/file27797851fa0347/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpUC1bK6/file27797851fa0347/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpUC1bK6/file27797851fa0347/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:02:01 2026 ----------
00:02:01 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpUC1bK6/file27797817346022/sample1_realign2transcript.bam', '/tmp/RtmpUC1bK6/file27797817346022/sample2_realign2transcript.bam', '/tmp/RtmpUC1bK6/file27797817346022/sample3_realign2transcript.bam'] /tmp/RtmpUC1bK6/file27797817346022/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 866, 'not_enough_coverage': 30, 'unmapped': 4})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797841a01e0e/config_file_2587000.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 @ Sat May  9 00:02:01 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpUC1bK6/file27797841a01e0e/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpUC1bK6/file27797841a01e0e/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpUC1bK6/file27797841a01e0e/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat May  9 00:02:03 2026 -------------
Inputs:  ['/tmp/RtmpUC1bK6/file27797851fa0347/sample1_realign2transcript.bam', '/tmp/RtmpUC1bK6/file27797851fa0347/sample2_realign2transcript.bam', '/tmp/RtmpUC1bK6/file27797851fa0347/sample3_realign2transcript.bam'] /tmp/RtmpUC1bK6/file27797851fa0347/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 863, 'not_enough_coverage': 32, 'unmapped': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:02:04 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpUC1bK6/file27797841a01e0e/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpUC1bK6/file27797841a01e0e/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpUC1bK6/file27797841a01e0e/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sat May  9 00:02:06 2026 ----------
2026-05-09T04:02:06.994780Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:02:06.995210Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797841a01e0e/sample1_realign2transcript.bam, contains 17 reference sequences.
2026-05-09T04:02:06.995220Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:02:06.995223Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:02:06.995323Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:02:06.995333Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-09T04:02:07.006205Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-09T04:02:07.006441Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3436  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:02:07.006477Z  INFO oarfish::bulk: Total number of alignment records : 513
2026-05-09T04:02:07.006480Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-09T04:02:07.006482Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-09T04:02:07.007392Z  INFO oarfish: oarfish completed successfully.
2026-05-09T04:02:07.015186Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:02:07.015593Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797841a01e0e/sample2_realign2transcript.bam, contains 17 reference sequences.
2026-05-09T04:02:07.015606Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:02:07.015609Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:02:07.015706Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:02:07.015716Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-09T04:02:07.026225Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-09T04:02:07.026489Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3402  │
│ aligned fraction too low        │ 8     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 292   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:02:07.026536Z  INFO oarfish::bulk: Total number of alignment records : 525
2026-05-09T04:02:07.026539Z  INFO oarfish::bulk: number of aligned reads : 292
2026-05-09T04:02:07.026541Z  INFO oarfish::bulk: number of unique alignments : 190
2026-05-09T04:02:07.027453Z  INFO oarfish: oarfish completed successfully.
2026-05-09T04:02:07.035255Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:02:07.035778Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797841a01e0e/sample3_realign2transcript.bam, contains 17 reference sequences.
2026-05-09T04:02:07.035792Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:02:07.035796Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:02:07.035887Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:02:07.035896Z  INFO oarfish: parsed reference information for 17 transcripts.
2026-05-09T04:02:07.046149Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-09T04:02:07.046393Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3342  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:02:07.046427Z  INFO oarfish::bulk: Total number of alignment records : 517
2026-05-09T04:02:07.046430Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-09T04:02:07.046432Z  INFO oarfish::bulk: number of unique alignments : 192
2026-05-09T04:02:07.047339Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779782f63558f/config_file_2587000.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 @ Sat May  9 00:02:07 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpUC1bK6/file2779782f63558f/sample1_align2genome.bam
sample2 ->/tmp/RtmpUC1bK6/file2779782f63558f/sample2_align2genome.bam
sample3 ->/tmp/RtmpUC1bK6/file2779782f63558f/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat May  9 00:02:29 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:02:30 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpUC1bK6/file2779782f63558f/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpUC1bK6/file2779782f63558f/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpUC1bK6/file2779782f63558f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:02:50 2026 ----------
2026-05-09T04:02:50.908215Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:02:50.908798Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779782f63558f/sample1_realign2transcript.bam, contains 15 reference sequences.
2026-05-09T04:02:50.908810Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:02:50.908814Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:02:50.908898Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:02:50.908907Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-09T04:02:50.918788Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-09T04:02:50.919028Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 3045  │
│ aligned fraction too low        │ 7     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 293   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:02:50.919060Z  INFO oarfish::bulk: Total number of alignment records : 495
2026-05-09T04:02:50.919064Z  INFO oarfish::bulk: number of aligned reads : 293
2026-05-09T04:02:50.919067Z  INFO oarfish::bulk: number of unique alignments : 208
2026-05-09T04:02:50.919989Z  INFO oarfish: oarfish completed successfully.
2026-05-09T04:02:50.930134Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:02:50.930690Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779782f63558f/sample2_realign2transcript.bam, contains 15 reference sequences.
2026-05-09T04:02:50.930705Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:02:50.930709Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:02:50.930795Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:02:50.930818Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-09T04:02:50.940609Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-09T04:02:50.940846Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2896  │
│ aligned fraction too low        │ 10    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 290   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:02:50.940876Z  INFO oarfish::bulk: Total number of alignment records : 511
2026-05-09T04:02:50.940879Z  INFO oarfish::bulk: number of aligned reads : 290
2026-05-09T04:02:50.940881Z  INFO oarfish::bulk: number of unique alignments : 198
2026-05-09T04:02:50.941807Z  INFO oarfish: oarfish completed successfully.
2026-05-09T04:02:50.951258Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:02:50.951774Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779782f63558f/sample3_realign2transcript.bam, contains 15 reference sequences.
2026-05-09T04:02:50.951786Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:02:50.951789Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:02:50.951873Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:02:50.951883Z  INFO oarfish: parsed reference information for 15 transcripts.
2026-05-09T04:02:50.961194Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-05-09T04:02:50.961411Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 2880  │
│ aligned fraction too low        │ 9     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 291   │
╰─────────────────────────────────┴───────╯

2026-05-09T04:02:50.961441Z  INFO oarfish::bulk: Total number of alignment records : 496
2026-05-09T04:02:50.961444Z  INFO oarfish::bulk: number of aligned reads : 291
2026-05-09T04:02:50.961446Z  INFO oarfish::bulk: number of unique alignments : 206
2026-05-09T04:02:50.962319Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779789cad417/config_file_2587000.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 @ Sat May  9 00:02:51 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpUC1bK6/file2779789cad417/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpUC1bK6/file2779789cad417/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpUC1bK6/file2779789cad417/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat May  9 00:02:53 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:02:53 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpUC1bK6/file2779789cad417/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpUC1bK6/file2779789cad417/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpUC1bK6/file2779789cad417/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat May  9 00:02:55 2026 ----------
00:02:55 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797814027ef3/config_file_2587000.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 @ Sat May  9 00:02:56 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpUC1bK6/file27797814027ef3/sample1_align2genome.bam
sample2 ->/tmp/RtmpUC1bK6/file27797814027ef3/sample2_align2genome.bam
sample3 ->/tmp/RtmpUC1bK6/file27797814027ef3/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat May  9 00:03:17 2026 -------------
Inputs:  ['/tmp/RtmpUC1bK6/file2779789cad417/sample1_realign2transcript.bam', '/tmp/RtmpUC1bK6/file2779789cad417/sample2_realign2transcript.bam', '/tmp/RtmpUC1bK6/file2779789cad417/sample3_realign2transcript.bam'] /tmp/RtmpUC1bK6/file2779789cad417/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:03:18 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpUC1bK6/file27797814027ef3/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpUC1bK6/file27797814027ef3/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpUC1bK6/file27797814027ef3/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:03:38 2026 ----------
00:03:38 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779785c02d9ab/config_file_2587000.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 @ Sat May  9 00:03:40 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779785c02d9ab/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:03:40 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpUC1bK6/file2779785c02d9ab/matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779785c02d9ab/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat May  9 00:03:40 2026 ----------------
00:03:40 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779785c02d9ab/align2genome.bam'
Inputs:  ['/tmp/RtmpUC1bK6/file27797814027ef3/sample1_realign2transcript.bam', '/tmp/RtmpUC1bK6/file27797814027ef3/sample2_realign2transcript.bam', '/tmp/RtmpUC1bK6/file27797814027ef3/sample3_realign2transcript.bam'] /tmp/RtmpUC1bK6/file27797814027ef3/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 895, 'not_enough_coverage': 5})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.59gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.58gene_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, 415458.62Read/s]
-- Running step: isoform_identification @ Sat May  9 00:03:42 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:03:55 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779785c02d9ab/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779785c02d9ab/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpUC1bK6/file2779785c02d9ab/matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779785c02d9ab/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 @ Sat May  9 00:03:55 2026 ----------
2026-05-09T04:03:55.262559Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:03:55.263111Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779785c02d9ab/realign2transcript.bam, contains 5 reference sequences.
2026-05-09T04:03:55.263138Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:03:55.263142Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:03:55.263207Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:03:55.263213Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-09T04:03:55.270292Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file277978255e010e/config_file_2587000.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 @ Sat May  9 00:03:55 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file277978255e010e/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:03:56 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpUC1bK6/file277978255e010e/matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file277978255e010e/align2genome.bam
-- Running step: gene_quantification @ Sat May  9 00:04:14 2026 ----------------
00:04:15 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file277978255e010e/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.42gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.41gene_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, 409264.28Read/s]
-- Running step: isoform_identification @ Sat May  9 00:04:15 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:04:26 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file277978255e010e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file277978255e010e/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpUC1bK6/file277978255e010e/matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file277978255e010e/realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:04:46 2026 ----------
2026-05-09T04:04:46.031321Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:04:46.031776Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file277978255e010e/realign2transcript.bam, contains 5 reference sequences.
2026-05-09T04:04:46.031790Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:04:46.031794Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:04:46.031854Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:04:46.031860Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-05-09T04:04:46.038260Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779787db0ec40/config_file_2587000.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 @ Sat May  9 00:04:46 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779787db0ec40/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:04:46 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpUC1bK6/file2779787db0ec40/matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779787db0ec40/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat May  9 00:04:47 2026 ----------------
00:04:47 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779787db0ec40/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.42gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 347994.16Read/s]
-- Running step: isoform_identification @ Sat May  9 00:04:47 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:04:59 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779787db0ec40/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779787db0ec40/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpUC1bK6/file2779787db0ec40/matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779787db0ec40/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 @ Sat May  9 00:04:59 2026 ----------
00:04:59 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779782387902f/config_file_2587000.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 @ Sat May  9 00:05:00 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779782387902f/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:05:00 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpUC1bK6/file2779782387902f/matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file2779782387902f/align2genome.bam
-- Running step: gene_quantification @ Sat May  9 00:05:19 2026 ----------------
00:05:19 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779782387902f/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.98gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.98gene_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, 411739.11Read/s]
-- Running step: isoform_identification @ Sat May  9 00:05:20 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:05:30 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779782387902f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779782387902f/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpUC1bK6/file2779782387902f/matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file2779782387902f/realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:05:48 2026 ----------
00:05:48 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779787432bd66/config_file_2587000.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 @ Sat May  9 00:05:49 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779787432bd66/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:05:49 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpUC1bK6/file2779787432bd66/matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779787432bd66/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat May  9 00:05:50 2026 ----------------
00:05:50 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779787432bd66/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.44gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.43gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 351423.02Read/s]
-- Running step: isoform_identification @ Sat May  9 00:05:50 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:05:51 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779787432bd66/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779787432bd66/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpUC1bK6/file2779787432bd66/matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779787432bd66/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 @ Sat May  9 00:05:51 2026 ----------
2026-05-09T04:05:51.631457Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:05:51.631883Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779787432bd66/realign2transcript.bam, contains 10 reference sequences.
2026-05-09T04:05:51.631893Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:05:51.631896Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:05:51.631969Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:05:51.631977Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-09T04:05:51.641387Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797828e1451d/config_file_2587000.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 @ Sat May  9 00:05:52 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file27797828e1451d/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:05:52 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797828e1451d/matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797828e1451d/align2genome.bam
-- Running step: gene_quantification @ Sat May  9 00:06:11 2026 ----------------
00:06:11 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file27797828e1451d/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.44gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.43gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 359285.93Read/s]
-- Running step: isoform_identification @ Sat May  9 00:06:11 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:06:12 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797828e1451d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797828e1451d/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797828e1451d/matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797828e1451d/realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:06:30 2026 ----------
2026-05-09T04:06:30.464248Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:06:30.464698Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797828e1451d/realign2transcript.bam, contains 10 reference sequences.
2026-05-09T04:06:30.464711Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:06:30.464715Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:06:30.464784Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:06:30.464792Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-05-09T04:06:30.474328Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779782dacbb35/config_file_2587000.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 @ Sat May  9 00:06:31 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779782dacbb35/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:06:31 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpUC1bK6/file2779782dacbb35/matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779782dacbb35/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat May  9 00:06:31 2026 ----------------
00:06:31 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779782dacbb35/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.29gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.29gene_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, 363193.52Read/s]
-- Running step: isoform_identification @ Sat May  9 00:06:32 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:06:32 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779782dacbb35/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779782dacbb35/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpUC1bK6/file2779782dacbb35/matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779782dacbb35/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 @ Sat May  9 00:06:33 2026 ----------
00:06:33 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779786644737b/config_file_2587000.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 @ Sat May  9 00:06:34 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779786644737b/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 8
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:06:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpUC1bK6/file2779786644737b/matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file2779786644737b/align2genome.bam
-- Running step: gene_quantification @ Sat May  9 00:06:53 2026 ----------------
00:06:53 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779786644737b/align2genome.bam'
	Counter({'counted_reads': 358})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.33gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.32gene_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, 365752.56Read/s]
-- Running step: isoform_identification @ Sat May  9 00:06:53 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:06:54 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779786644737b/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779786644737b/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpUC1bK6/file2779786644737b/matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file2779786644737b/realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:07:11 2026 ----------
00:07:11 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779787b1ea0b2/config_file_2587000.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 @ Sat May  9 00:07:13 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779787b1ea0b2/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 927
Number of chimera reads: 3
All done!
Reads	Barcodes
26	1
23	3
22	1
21	3
20	1
19	1
18	1
16	2
15	1
14	2
13	3
12	5
11	2
10	6
9	9
8	3
7	6
6	11
5	8
4	8
3	39
2	19
1	2
Loading known barcodes from /tmp/RtmpUC1bK6/file2779787b1ea0b2/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	5
6	2
5	7
4	5
3	19
2	27
1	53
Loading known barcodes from /tmp/RtmpUC1bK6/file2779787b1ea0b2/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 279
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
7	5
6	4
5	1
4	15
3	12
2	23
1	65
Loading known barcodes from /tmp/RtmpUC1bK6/file2779787b1ea0b2/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:07:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat May  9 00:07:18 2026 ----------------
00:07:18 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_align2genome.bam',
'/tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_align2genome.bam',
'/tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_align2genome.bam', and
'/tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 183963.93Read/s]
parsing /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.51gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 580125.03Read/s]
parsing /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 570312.19Read/s]
parsing /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.20gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.19gene_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, 460002.63Read/s]
-- Running step: isoform_identification @ Sat May  9 00:07:19 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 7 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:07:42 2026 -------------------
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq, /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample1.fq.gz, /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample2.fq.gz, /tmp/RtmpUC1bK6/file2779787b1ea0b2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat May  9 00:07:44 2026 ----------
2026-05-09T04:07:44.819343Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:07:44.819724Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779787b1ea0b2/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-09T04:07:44.819736Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:07:44.819740Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:07:44.819802Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:07:44.819807Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-09T04:07:44.830493Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:07:45.135055Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:07:45.135479Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-09T04:07:45.135491Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:07:45.135495Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:07:45.135556Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:07:45.135562Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-09T04:07:45.140808Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:07:45.421263Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:07:45.421765Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-09T04:07:45.421777Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:07:45.421780Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:07:45.421836Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:07:45.421842Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-09T04:07:45.426992Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:07:45.707925Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:07:45.708293Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779787b1ea0b2/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-09T04:07:45.708302Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:07:45.708305Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:07:45.708368Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:07:45.708380Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-09T04:07:45.714013Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797833425bc9/config_file_2587000.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 @ Sat May  9 00:07:46 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file27797833425bc9/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 930
Number of chimera reads: 3
All done!
Reads	Barcodes
28	1
25	1
23	3
22	1
21	1
19	2
18	1
17	1
16	2
15	2
14	2
13	4
12	2
11	5
10	4
9	7
8	7
7	2
6	11
5	12
4	7
3	37
2	20
1	2
Loading known barcodes from /tmp/RtmpUC1bK6/file27797833425bc9/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 284
Number of chimera reads: 1
All done!
Reads	Barcodes
10	1
9	1
8	3
7	2
6	4
5	7
4	6
3	15
2	21
1	60
Loading known barcodes from /tmp/RtmpUC1bK6/file27797833425bc9/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 1
All done!
Reads	Barcodes
8	2
7	3
6	1
5	6
4	9
3	19
2	29
1	56
Loading known barcodes from /tmp/RtmpUC1bK6/file27797833425bc9/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:07:47 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797833425bc9/sampleA_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797833425bc9/sampleA_align2genome.bam
/tmp/RtmpUC1bK6/file27797833425bc9/sample1_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797833425bc9/sample1_align2genome.bam
/tmp/RtmpUC1bK6/file27797833425bc9/sample2_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797833425bc9/sample2_align2genome.bam
/tmp/RtmpUC1bK6/file27797833425bc9/sample3_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797833425bc9/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat May  9 00:08:09 2026 ----------------
00:08:09 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file27797833425bc9/sampleA_align2genome.bam',
'/tmp/RtmpUC1bK6/file27797833425bc9/sample1_align2genome.bam',
'/tmp/RtmpUC1bK6/file27797833425bc9/sample2_align2genome.bam', and
'/tmp/RtmpUC1bK6/file27797833425bc9/sample3_align2genome.bam'
parsing /tmp/RtmpUC1bK6/file27797833425bc9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.05gene_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, 157533.73Read/s]
parsing /tmp/RtmpUC1bK6/file27797833425bc9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.99gene_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, 504123.08Read/s]
parsing /tmp/RtmpUC1bK6/file27797833425bc9/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.49gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 539002.78Read/s]
parsing /tmp/RtmpUC1bK6/file27797833425bc9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.06gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 414981.80Read/s]
-- Running step: isoform_identification @ Sat May  9 00:08:10 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 8 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:08:34 2026 -------------------
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797833425bc9/fastq, /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample1.fq.gz, /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample2.fq.gz, /tmp/RtmpUC1bK6/file27797833425bc9/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797833425bc9/sampleA_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797833425bc9/sample1_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797833425bc9/sample2_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797833425bc9/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797833425bc9/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797833425bc9/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797833425bc9/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797833425bc9/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797833425bc9/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797833425bc9/sampleA_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797833425bc9/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797833425bc9/sample1_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797833425bc9/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797833425bc9/sample2_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797833425bc9/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797833425bc9/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:08:57 2026 ----------
2026-05-09T04:08:57.086129Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:08:57.086496Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797833425bc9/sampleA_realign2transcript.bam, contains 6 reference sequences.
2026-05-09T04:08:57.086509Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:08:57.086513Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:08:57.086574Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:08:57.086582Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-09T04:08:57.097597Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:08:57.452321Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:08:57.452688Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797833425bc9/sample1_realign2transcript.bam, contains 6 reference sequences.
2026-05-09T04:08:57.452701Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:08:57.452705Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:08:57.452764Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:08:57.452771Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-09T04:08:57.458447Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:08:57.773980Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:08:57.774358Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797833425bc9/sample2_realign2transcript.bam, contains 6 reference sequences.
2026-05-09T04:08:57.774368Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:08:57.774381Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:08:57.774438Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:08:57.774445Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-09T04:08:57.779680Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:08:58.114296Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:08:58.114667Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797833425bc9/sample3_realign2transcript.bam, contains 6 reference sequences.
2026-05-09T04:08:58.114679Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:08:58.114682Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:08:58.114742Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:08:58.114748Z  INFO oarfish: parsed reference information for 6 transcripts.
2026-05-09T04:08:58.120558Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779782ceffe0a/config_file_2587000.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 @ Sat May  9 00:08:58 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779782ceffe0a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 929
Number of chimera reads: 2
All done!
Reads	Barcodes
27	1
26	1
24	1
22	2
21	1
20	3
19	1
18	1
17	2
16	2
15	2
14	1
12	1
11	7
10	5
9	5
8	9
7	5
6	13
5	11
4	5
3	33
2	22
1	3
Loading known barcodes from /tmp/RtmpUC1bK6/file2779782ceffe0a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 281
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	3
7	2
6	5
5	3
4	9
3	13
2	31
1	55
Loading known barcodes from /tmp/RtmpUC1bK6/file2779782ceffe0a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 0
All done!
Reads	Barcodes
9	1
8	3
7	1
6	5
5	5
4	6
3	18
2	30
1	50
Loading known barcodes from /tmp/RtmpUC1bK6/file2779782ceffe0a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:08:59 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779782ceffe0a/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/RtmpUC1bK6/file2779782ceffe0a/sample1_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779782ceffe0a/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/RtmpUC1bK6/file2779782ceffe0a/sample2_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779782ceffe0a/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/RtmpUC1bK6/file2779782ceffe0a/sample3_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779782ceffe0a/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 @ Sat May  9 00:09:03 2026 ----------------
00:09:03 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_align2genome.bam',
'/tmp/RtmpUC1bK6/file2779782ceffe0a/sample1_align2genome.bam',
'/tmp/RtmpUC1bK6/file2779782ceffe0a/sample2_align2genome.bam', and
'/tmp/RtmpUC1bK6/file2779782ceffe0a/sample3_align2genome.bam'
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.23gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 162167.65Read/s]
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 507637.49Read/s]
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 519302.69Read/s]
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 379712.48Read/s]
-- Running step: isoform_identification @ Sat May  9 00:09:04 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
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Detected 8 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:09:27 2026 -------------------
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq, /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample1.fq.gz, /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample2.fq.gz, /tmp/RtmpUC1bK6/file2779782ceffe0a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779782ceffe0a/sample1_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779782ceffe0a/sample2_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779782ceffe0a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779782ceffe0a/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779782ceffe0a/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779782ceffe0a/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779782ceffe0a/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/RtmpUC1bK6/file2779782ceffe0a/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779782ceffe0a/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/RtmpUC1bK6/file2779782ceffe0a/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779782ceffe0a/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/RtmpUC1bK6/file2779782ceffe0a/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779782ceffe0a/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 @ Sat May  9 00:09:29 2026 ----------
00:09:29 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file2779782ceffe0a/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample1_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file2779782ceffe0a/sample1_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample2_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file2779782ceffe0a/sample2_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample3_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file2779782ceffe0a/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file2779782ceffe0a/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797823837504/config_file_2587000.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 @ Sat May  9 00:09:31 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file27797823837504/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797823837504/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file27797823837504/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file27797823837504/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 924
Number of chimera reads: 2
All done!
Reads	Barcodes
26	1
25	1
22	1
21	3
20	2
19	2
18	1
17	4
16	1
15	1
14	1
13	1
12	1
11	7
10	5
9	3
8	11
7	3
6	5
5	19
4	7
3	35
2	19
1	3
Loading known barcodes from /tmp/RtmpUC1bK6/file27797823837504/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797823837504/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 279
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	3
6	5
5	8
4	7
3	11
2	23
1	66
Loading known barcodes from /tmp/RtmpUC1bK6/file27797823837504/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797823837504/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 277
Number of chimera reads: 0
All done!
Reads	Barcodes
8	1
7	3
6	8
5	5
4	4
3	19
2	22
1	60
Loading known barcodes from /tmp/RtmpUC1bK6/file27797823837504/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797823837504/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:09:32 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797823837504/sampleA_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797823837504/sampleA_align2genome.bam
/tmp/RtmpUC1bK6/file27797823837504/sample1_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797823837504/sample1_align2genome.bam
/tmp/RtmpUC1bK6/file27797823837504/sample2_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797823837504/sample2_align2genome.bam
/tmp/RtmpUC1bK6/file27797823837504/sample3_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797823837504/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat May  9 00:09:54 2026 ----------------
00:09:54 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file27797823837504/sampleA_align2genome.bam',
'/tmp/RtmpUC1bK6/file27797823837504/sample1_align2genome.bam',
'/tmp/RtmpUC1bK6/file27797823837504/sample2_align2genome.bam', and
'/tmp/RtmpUC1bK6/file27797823837504/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 5})
	Counter({'counted_reads': 262, 'not_enough_coverage': 8, 'unmapped': 2})
	Counter({'counted_reads': 264, 'not_enough_coverage': 8, 'unmapped': 1})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
parsing /tmp/RtmpUC1bK6/file27797823837504/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.87gene_group/s]
/home/biocbuild/bbs-3.24-bioc/R/site-library/FLAMES/python/count_gene.py:704: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 133574.86Read/s]
parsing /tmp/RtmpUC1bK6/file27797823837504/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.96gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 485406.91Read/s]
parsing /tmp/RtmpUC1bK6/file27797823837504/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.24gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.23gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 514689.05Read/s]
parsing /tmp/RtmpUC1bK6/file27797823837504/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.87gene_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, 394038.56Read/s]
-- Running step: isoform_identification @ Sat May  9 00:09:55 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 8 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat May  9 00:10:18 2026 -------------------
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797823837504/fastq, /tmp/RtmpUC1bK6/file27797823837504/fastq/sample1.fq.gz, /tmp/RtmpUC1bK6/file27797823837504/fastq/sample2.fq.gz, /tmp/RtmpUC1bK6/file27797823837504/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797823837504/sampleA_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797823837504/sample1_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797823837504/sample2_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797823837504/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797823837504/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797823837504/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797823837504/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797823837504/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797823837504/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797823837504/sampleA_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797823837504/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797823837504/sample1_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797823837504/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797823837504/sample2_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797823837504/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797823837504/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:10:39 2026 ----------
00:10:39 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpUC1bK6/file27797823837504/sampleA_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file27797823837504/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file27797823837504/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file27797823837504/sample1_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file27797823837504/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file27797823837504/sample1_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file27797823837504/sample2_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file27797823837504/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file27797823837504/sample2_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file27797823837504/sample3_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file27797823837504/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file27797823837504/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779784257185a/config_file_2587000.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 @ Sat May  9 00:10:41 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779784257185a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 928
Number of chimera reads: 2
All done!
Reads	Barcodes
28	1
26	1
21	2
20	3
19	4
17	2
14	4
13	2
12	5
11	7
10	1
9	6
8	4
7	7
6	12
5	15
4	4
3	35
2	19
1	3
Loading known barcodes from /tmp/RtmpUC1bK6/file2779784257185a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	4
6	3
5	5
4	8
3	12
2	36
1	54
Loading known barcodes from /tmp/RtmpUC1bK6/file2779784257185a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 282
Number of chimera reads: 0
All done!
Reads	Barcodes
9	2
7	1
6	4
5	8
4	14
3	9
2	27
1	59
Loading known barcodes from /tmp/RtmpUC1bK6/file2779784257185a/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:10:42 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpUC1bK6/file2779784257185a/sampleA_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779784257185a/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpUC1bK6/file2779784257185a/sample1_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779784257185a/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpUC1bK6/file2779784257185a/sample2_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779784257185a/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpUC1bK6/file2779784257185a/sample3_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779784257185a/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat May  9 00:10:45 2026 ----------------
00:10:45 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779784257185a/sampleA_align2genome.bam',
'/tmp/RtmpUC1bK6/file2779784257185a/sample1_align2genome.bam',
'/tmp/RtmpUC1bK6/file2779784257185a/sample2_align2genome.bam', and
'/tmp/RtmpUC1bK6/file2779784257185a/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 5})
	Counter({'counted_reads': 265, 'not_enough_coverage': 8, 'unmapped': 2})
	Counter({'counted_reads': 261, 'not_enough_coverage': 9, 'unmapped': 1})
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
parsing /tmp/RtmpUC1bK6/file2779784257185a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 166342.94Read/s]
parsing /tmp/RtmpUC1bK6/file2779784257185a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.70gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 518506.65Read/s]
parsing /tmp/RtmpUC1bK6/file2779784257185a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.08gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 508326.55Read/s]
parsing /tmp/RtmpUC1bK6/file2779784257185a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.22gene_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, 431317.51Read/s]
-- Running step: isoform_identification @ Sat May  9 00:10:47 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:10:47 2026 -------------------
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779784257185a/fastq, /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample1.fq.gz, /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample2.fq.gz, /tmp/RtmpUC1bK6/file2779784257185a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779784257185a/sampleA_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779784257185a/sample1_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779784257185a/sample2_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779784257185a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779784257185a/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779784257185a/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779784257185a/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779784257185a/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpUC1bK6/file2779784257185a/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779784257185a/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpUC1bK6/file2779784257185a/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779784257185a/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpUC1bK6/file2779784257185a/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779784257185a/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpUC1bK6/file2779784257185a/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779784257185a/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat May  9 00:10:55 2026 ----------
2026-05-09T04:10:55.090015Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:10:55.090537Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779784257185a/sampleA_realign2transcript.bam, contains 27 reference sequences.
2026-05-09T04:10:55.090550Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:10:55.090554Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:10:55.090675Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:10:55.090689Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-09T04:10:55.133264Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:10:55.657099Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:10:55.657538Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779784257185a/sample1_realign2transcript.bam, contains 27 reference sequences.
2026-05-09T04:10:55.657550Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:10:55.657555Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:10:55.657675Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:10:55.657689Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-09T04:10:55.673586Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:10:56.240027Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:10:56.240634Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779784257185a/sample2_realign2transcript.bam, contains 27 reference sequences.
2026-05-09T04:10:56.242859Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:10:56.242867Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:10:56.242988Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:10:56.243003Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-09T04:10:56.258401Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:10:56.780797Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:10:56.781353Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file2779784257185a/sample3_realign2transcript.bam, contains 27 reference sequences.
2026-05-09T04:10:56.781364Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:10:56.781368Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:10:56.781495Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:10:56.781510Z  INFO oarfish: parsed reference information for 27 transcripts.
2026-05-09T04:10:56.800125Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797827e8e6b7/config_file_2587000.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 @ Sat May  9 00:10:57 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file27797827e8e6b7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 932
Number of chimera reads: 3
All done!
Reads	Barcodes
26	1
25	1
24	1
22	2
21	1
20	4
19	1
18	1
15	3
14	1
13	1
12	3
11	9
10	5
9	5
8	6
7	3
6	15
5	10
4	6
3	37
2	20
1	1
Loading known barcodes from /tmp/RtmpUC1bK6/file27797827e8e6b7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 1
All done!
Reads	Barcodes
8	2
7	4
6	5
5	2
4	9
3	18
2	27
1	56
Loading known barcodes from /tmp/RtmpUC1bK6/file27797827e8e6b7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 284
Number of chimera reads: 1
All done!
Reads	Barcodes
8	3
7	4
6	4
5	3
4	9
3	17
2	26
1	58
Loading known barcodes from /tmp/RtmpUC1bK6/file27797827e8e6b7/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:10:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_align2genome.bam
/tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_align2genome.bam
/tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_align2genome.bam
/tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat May  9 00:11:20 2026 ----------------
00:11:20 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_align2genome.bam',
'/tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_align2genome.bam',
'/tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_align2genome.bam', and
'/tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_align2genome.bam'
parsing /tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.99gene_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, 135083.99Read/s]
parsing /tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.08gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 480337.15Read/s]
parsing /tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 27.94gene_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, 486127.03Read/s]
parsing /tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 386885.58Read/s]
-- Running step: isoform_identification @ Sat May  9 00:11:21 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:11:22 2026 -------------------
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq, /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample1.fq.gz, /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample2.fq.gz, /tmp/RtmpUC1bK6/file27797827e8e6b7/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:11:49 2026 ----------
2026-05-09T04:11:49.261332Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:11:49.261975Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797827e8e6b7/sampleA_realign2transcript.bam, contains 28 reference sequences.
2026-05-09T04:11:49.262000Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:11:49.262004Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:11:49.262130Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:11:49.262143Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-09T04:11:49.304558Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:11:50.038538Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:11:50.038955Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797827e8e6b7/sample1_realign2transcript.bam, contains 28 reference sequences.
2026-05-09T04:11:50.038967Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:11:50.038971Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:11:50.039092Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:11:50.039106Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-09T04:11:50.054370Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:11:50.631254Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:11:50.631651Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797827e8e6b7/sample2_realign2transcript.bam, contains 28 reference sequences.
2026-05-09T04:11:50.631664Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:11:50.631668Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:11:50.631788Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:11:50.631803Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-09T04:11:50.647653Z  INFO oarfish::single_cell: Processed 100 cells.
2026-05-09T04:11:51.201096Z  INFO oarfish: setting user-provided filter parameters.
2026-05-09T04:11:51.201492Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpUC1bK6/file27797827e8e6b7/sample3_realign2transcript.bam, contains 28 reference sequences.
2026-05-09T04:11:51.201503Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-05-09T04:11:51.201507Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-05-09T04:11:51.201635Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-05-09T04:11:51.201648Z  INFO oarfish: parsed reference information for 28 transcripts.
2026-05-09T04:11:51.220185Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file2779786b5043d8/config_file_2587000.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 @ Sat May  9 00:11:52 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file2779786b5043d8/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 928
Number of chimera reads: 2
All done!
Reads	Barcodes
26	1
23	2
22	1
21	1
20	2
19	2
18	4
16	2
15	1
14	1
13	1
12	5
11	6
10	5
9	4
8	7
7	3
6	15
5	11
4	2
3	35
2	23
1	3
Loading known barcodes from /tmp/RtmpUC1bK6/file2779786b5043d8/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 278
Number of chimera reads: 0
All done!
Reads	Barcodes
7	4
6	3
5	9
4	11
3	13
2	23
1	59
Loading known barcodes from /tmp/RtmpUC1bK6/file2779786b5043d8/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 282
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	1
7	5
6	2
5	8
4	7
3	14
2	32
1	46
Loading known barcodes from /tmp/RtmpUC1bK6/file2779786b5043d8/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:11:53 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779786b5043d8/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/RtmpUC1bK6/file2779786b5043d8/sample1_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779786b5043d8/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/RtmpUC1bK6/file2779786b5043d8/sample2_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779786b5043d8/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/RtmpUC1bK6/file2779786b5043d8/sample3_matched_reads.fastq.gz -> /tmp/RtmpUC1bK6/file2779786b5043d8/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 @ Sat May  9 00:11:56 2026 ----------------
00:11:56 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_align2genome.bam',
'/tmp/RtmpUC1bK6/file2779786b5043d8/sample1_align2genome.bam',
'/tmp/RtmpUC1bK6/file2779786b5043d8/sample2_align2genome.bam', and
'/tmp/RtmpUC1bK6/file2779786b5043d8/sample3_align2genome.bam'
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.60gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 152588.95Read/s]
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  5.14gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  5.13gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 508228.00Read/s]
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 26.80gene_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, 471736.55Read/s]
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.26gene_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, 407356.36Read/s]
-- Running step: isoform_identification @ Sat May  9 00:11:58 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:11:58 2026 -------------------
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779786b5043d8/fastq, /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample1.fq.gz, /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample2.fq.gz, /tmp/RtmpUC1bK6/file2779786b5043d8/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779786b5043d8/sample1_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779786b5043d8/sample2_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file2779786b5043d8/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779786b5043d8/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779786b5043d8/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file2779786b5043d8/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779786b5043d8/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/RtmpUC1bK6/file2779786b5043d8/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779786b5043d8/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/RtmpUC1bK6/file2779786b5043d8/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779786b5043d8/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/RtmpUC1bK6/file2779786b5043d8/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpUC1bK6/file2779786b5043d8/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 @ Sat May  9 00:12:01 2026 ----------
00:12:01 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file2779786b5043d8/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample1_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file2779786b5043d8/sample1_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample2_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file2779786b5043d8/sample2_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample3_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file2779786b5043d8/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file2779786b5043d8/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
i Writing configuration to: /tmp/RtmpUC1bK6/file27797820856224/config_file_2587000.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 @ Sat May  9 00:12:04 2026 ----------------
Using flexiplex for barcode demultiplexing.
Loading known barcodes from /tmp/RtmpUC1bK6/file27797820856224/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797820856224/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file27797820856224/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpUC1bK6/file27797820856224/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 993
Number of reads where at least one barcode was found: 929
Number of chimera reads: 2
All done!
Reads	Barcodes
29	1
24	1
22	1
21	2
20	3
19	1
18	2
16	4
15	1
14	1
13	5
11	4
10	7
9	5
8	9
7	3
6	13
5	14
4	2
3	32
2	20
1	6
Loading known barcodes from /tmp/RtmpUC1bK6/file27797820856224/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797820856224/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 281
Number of chimera reads: 1
All done!
Reads	Barcodes
9	1
8	3
7	1
6	4
5	5
4	8
3	16
2	33
1	50
Loading known barcodes from /tmp/RtmpUC1bK6/file27797820856224/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797820856224/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 300
Number of reads where at least one barcode was found: 280
Number of chimera reads: 0
All done!
Reads	Barcodes
10	1
9	1
7	1
6	4
5	10
4	7
3	16
2	28
1	51
Loading known barcodes from /tmp/RtmpUC1bK6/file27797820856224/bc_allow.tsv
Number of known barcodes: 143
FLEXIPLEX 1.02.6
Setting max flanking sequence edit distance to 8
Setting number of threads to 1
Search pattern:
primer: CTACACGACGCTCTTCCGATCT
CB: NNNNNNNNNNNNNNNN
UB: NNNNNNNNNNNN
polyT: TTTTTTTTT
CB:Z: tag field: CB
Processing file: /tmp/RtmpUC1bK6/file27797820856224/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sat May  9 00:12:06 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797820856224/sampleA_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797820856224/sampleA_align2genome.bam
/tmp/RtmpUC1bK6/file27797820856224/sample1_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797820856224/sample1_align2genome.bam
/tmp/RtmpUC1bK6/file27797820856224/sample2_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797820856224/sample2_align2genome.bam
/tmp/RtmpUC1bK6/file27797820856224/sample3_matched_reads.fastq.gz ->/tmp/RtmpUC1bK6/file27797820856224/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat May  9 00:12:28 2026 ----------------
00:12:28 Sat May 09 2026 quantify genes 
Using BAM(s): '/tmp/RtmpUC1bK6/file27797820856224/sampleA_align2genome.bam',
'/tmp/RtmpUC1bK6/file27797820856224/sample1_align2genome.bam',
'/tmp/RtmpUC1bK6/file27797820856224/sample2_align2genome.bam', and
'/tmp/RtmpUC1bK6/file27797820856224/sample3_align2genome.bam'
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 275})
	Counter({'counted_reads': 275, 'not_enough_coverage': 1})
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
parsing /tmp/RtmpUC1bK6/file27797820856224/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 14.31gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 144941.05Read/s]
parsing /tmp/RtmpUC1bK6/file27797820856224/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 29.81gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 460993.58Read/s]
parsing /tmp/RtmpUC1bK6/file27797820856224/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 28.41gene_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, 507293.66Read/s]
parsing /tmp/RtmpUC1bK6/file27797820856224/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.03gene_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, 399457.52Read/s]
-- Running step: isoform_identification @ Sat May  9 00:12:29 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat May  9 00:12:30 2026 -------------------
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797820856224/fastq, /tmp/RtmpUC1bK6/file27797820856224/fastq/sample1.fq.gz, /tmp/RtmpUC1bK6/file27797820856224/fastq/sample2.fq.gz, /tmp/RtmpUC1bK6/file27797820856224/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797820856224/sampleA_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797820856224/sample1_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797820856224/sample2_matched_reads.fastq.gz, /tmp/RtmpUC1bK6/file27797820856224/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpUC1bK6/file27797820856224/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797820856224/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797820856224/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpUC1bK6/file27797820856224/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpUC1bK6/file27797820856224/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797820856224/sampleA_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797820856224/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797820856224/sample1_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797820856224/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797820856224/sample2_realign2transcript.bam
/tmp/RtmpUC1bK6/file27797820856224/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpUC1bK6/file27797820856224/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat May  9 00:12:51 2026 ----------
00:12:51 Sat May 09 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpUC1bK6/file27797820856224/sampleA_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file27797820856224/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file27797820856224/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file27797820856224/sample1_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file27797820856224/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file27797820856224/sample1_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file27797820856224/sample2_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file27797820856224/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file27797820856224/sample2_realign2transcript.bamdone
parsing /tmp/RtmpUC1bK6/file27797820856224/sample3_realign2transcript.bam...
parsing /tmp/RtmpUC1bK6/file27797820856224/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpUC1bK6/file27797820856224/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Filtering for genes with at least 2 detected isoforms ...
	6 isoform(s) left.

Aggregating counts by cluster labels ...

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

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

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

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

[ FAIL 0 | WARN 182 | SKIP 0 | PASS 98 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 275})
	Counter({'counted_reads': 272})
	Counter({'counted_reads': 358})
> 
> proc.time()
   user  system elapsed 
828.868  55.220 865.102