##############################################################################
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###
### Running command:
###
###   /home/biocbuild/bbs-3.23-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.23-bioc/R/site-library --timings FLAMES_2.5.1.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck’
* using R Under development (unstable) (2025-10-20 r88955)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.5.1’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... OK
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     25.022  0.536  25.559
find_variants                21.698  0.708  21.790
blaze                         4.351 16.942  12.062
bulk_long_pipeline            2.344 12.584   2.523
sc_long_multisample_pipeline  8.150  6.065   8.011
sc_plot_genotype             11.345  0.107  10.287
MultiSampleSCPipeline        10.296  0.924  11.672
sc_DTU_analysis               7.103  2.265   7.096
plot_isoform_heatmap          7.286  0.220   7.524
create_sce_from_dir           3.680  2.824   3.884
sc_long_pipeline              3.166  2.226   2.837
* 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: 5 NOTEs
See
  ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.23-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


* installing to library ‘/home/biocbuild/bbs-3.23-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.5.1’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.23-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.23-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R Under development (unstable) (2025-10-20 r88955) -- "Unsuffered Consequences"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

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Type 'license()' or 'licence()' for distribution details.

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> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd16dd4cf7/config_file_3767229.json 
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd16dd4cf7/config_file_3767229.json 
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd16dd4cf7/config_file_3767229.json 
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd5938da73/config_file_3767229.json 
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd16a2acf8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd2511fbc0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd2511fbc0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd5294416/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd5294416/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd5294416/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd5294416/musc_rps24_4.fastq
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd7fcc122/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd26cfb705/config_file_3767229.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 @ Sun Nov 16 23:51:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp2P9shN/file397bbd26cfb705/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp2P9shN/file397bbd26cfb705/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp2P9shN/file397bbd26cfb705/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:51:21 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:51:28] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:51:28] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:51:28] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:51:28] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:51:30] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:51:30] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.


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

Detected 4 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 @ Sun Nov 16 23:51:45 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp2P9shN/file397bbd26cfb705/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmp2P9shN/file397bbd26cfb705/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmp2P9shN/file397bbd26cfb705/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sun Nov 16 23:51:46 2025 ----------
2025-11-17T04:51:46.203161Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:51:46.203673Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd26cfb705/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:51:46.203694Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:51:46.203727Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:51:46.203785Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:51:46.203797Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:51:46.205878Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:51:46.206052Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:51:46.206090Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-11-17T04:51:46.206104Z  INFO oarfish::bulk: number of aligned reads : 96
2025-11-17T04:51:46.206111Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-17T04:51:46.206720Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:51:46.215546Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:51:46.215971Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd26cfb705/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:51:46.215992Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:51:46.216000Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:51:46.216070Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:51:46.216081Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:51:46.217673Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:51:46.217804Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:51:46.217860Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-11-17T04:51:46.217868Z  INFO oarfish::bulk: number of aligned reads : 95
2025-11-17T04:51:46.217881Z  INFO oarfish::bulk: number of unique alignments : 82
2025-11-17T04:51:46.218612Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:51:46.226572Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:51:46.226974Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd26cfb705/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:51:46.227020Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:51:46.227028Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:51:46.227089Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:51:46.227102Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:51:46.229919Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-11-17T04:51:46.230099Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-11-17T04:51:46.230157Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-11-17T04:51:46.230165Z  INFO oarfish::bulk: number of aligned reads : 179
2025-11-17T04:51:46.230172Z  INFO oarfish::bulk: number of unique alignments : 143
2025-11-17T04:51:46.230906Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd7e2936f1/config_file_3767229.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 @ Sun Nov 16 23:51:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp2P9shN/file397bbd7e2936f1/sample1_align2genome.bam
sample2 ->/tmp/Rtmp2P9shN/file397bbd7e2936f1/sample2_align2genome.bam
sample3 ->/tmp/Rtmp2P9shN/file397bbd7e2936f1/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:52:07 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 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 @ Sun Nov 16 23:52:26 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp2P9shN/file397bbd7e2936f1/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp2P9shN/file397bbd7e2936f1/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp2P9shN/file397bbd7e2936f1/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Nov 16 23:52:45 2025 ----------
2025-11-17T04:52:45.883784Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:52:45.884369Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd7e2936f1/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:52:45.884420Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:52:45.884428Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:52:45.884489Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:52:45.884500Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:52:45.886371Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:52:45.886529Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:52:45.886579Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-11-17T04:52:45.886586Z  INFO oarfish::bulk: number of aligned reads : 96
2025-11-17T04:52:45.886593Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-17T04:52:45.887281Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:52:45.898933Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:52:45.899304Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd7e2936f1/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:52:45.899321Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:52:45.899349Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:52:45.899403Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:52:45.899413Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:52:45.900958Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:52:45.901103Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:52:45.901148Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-11-17T04:52:45.901155Z  INFO oarfish::bulk: number of aligned reads : 95
2025-11-17T04:52:45.901161Z  INFO oarfish::bulk: number of unique alignments : 82
2025-11-17T04:52:45.901749Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:52:45.912913Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:52:45.913287Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd7e2936f1/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:52:45.913306Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:52:45.913314Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:52:45.913381Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:52:45.913393Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:52:45.916043Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-11-17T04:52:45.916197Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-11-17T04:52:45.916251Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-11-17T04:52:45.916259Z  INFO oarfish::bulk: number of aligned reads : 179
2025-11-17T04:52:45.916270Z  INFO oarfish::bulk: number of unique alignments : 143
2025-11-17T04:52:45.916973Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd15ac78a5/config_file_3767229.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 @ Sun Nov 16 23:52:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp2P9shN/file397bbd15ac78a5/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp2P9shN/file397bbd15ac78a5/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp2P9shN/file397bbd15ac78a5/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:52:47 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 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 @ Sun Nov 16 23:53:05 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp2P9shN/file397bbd15ac78a5/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmp2P9shN/file397bbd15ac78a5/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmp2P9shN/file397bbd15ac78a5/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sun Nov 16 23:53:06 2025 ----------
23:53:06 Sun Nov 16 2025 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)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd30b3fff3/config_file_3767229.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 @ Sun Nov 16 23:53:07 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample1_align2genome.bam
sample2 ->/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample2_align2genome.bam
sample3 ->/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:53:26 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 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 @ Sun Nov 16 23:53:44 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Nov 16 23:54:03 2025 ----------
23:54:03 Sun Nov 16 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmp2P9shN/file397bbd15ac78a5/sample1_realign2transcript.bam', '/tmp/Rtmp2P9shN/file397bbd15ac78a5/sample2_realign2transcript.bam', '/tmp/Rtmp2P9shN/file397bbd15ac78a5/sample3_realign2transcript.bam'] /tmp/Rtmp2P9shN/file397bbd15ac78a5/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd6de17125/config_file_3767229.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 @ Sun Nov 16 23:54:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp2P9shN/file397bbd6de17125/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp2P9shN/file397bbd6de17125/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp2P9shN/file397bbd6de17125/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:54:04 2025 -------------
Inputs:  ['/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample1_realign2transcript.bam', '/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample2_realign2transcript.bam', '/tmp/Rtmp2P9shN/file397bbd30b3fff3/sample3_realign2transcript.bam'] /tmp/Rtmp2P9shN/file397bbd30b3fff3/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Nov 16 23:54:05 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp2P9shN/file397bbd6de17125/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmp2P9shN/file397bbd6de17125/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmp2P9shN/file397bbd6de17125/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sun Nov 16 23:54:06 2025 ----------
2025-11-17T04:54:06.152497Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:54:06.152884Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd6de17125/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T04:54:06.152932Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:54:06.152940Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:54:06.153010Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:54:06.153023Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T04:54:06.155698Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:54:06.155835Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:54:06.155887Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-11-17T04:54:06.155895Z  INFO oarfish::bulk: number of aligned reads : 98
2025-11-17T04:54:06.155902Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-17T04:54:06.156520Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:54:06.163867Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:54:06.164234Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd6de17125/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T04:54:06.164252Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:54:06.164279Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:54:06.164346Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:54:06.164359Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T04:54:06.167067Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:54:06.167212Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:54:06.167255Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-11-17T04:54:06.167268Z  INFO oarfish::bulk: number of aligned reads : 97
2025-11-17T04:54:06.167274Z  INFO oarfish::bulk: number of unique alignments : 79
2025-11-17T04:54:06.167871Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:54:06.174872Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:54:06.175233Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd6de17125/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T04:54:06.175252Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:54:06.175259Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:54:06.175338Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:54:06.175351Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T04:54:06.179604Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:54:06.179757Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-11-17T04:54:06.179811Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-11-17T04:54:06.179819Z  INFO oarfish::bulk: number of aligned reads : 187
2025-11-17T04:54:06.179832Z  INFO oarfish::bulk: number of unique alignments : 140
2025-11-17T04:54:06.180562Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd448bd30a/config_file_3767229.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 @ Sun Nov 16 23:54:06 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp2P9shN/file397bbd448bd30a/sample1_align2genome.bam
sample2 ->/tmp/Rtmp2P9shN/file397bbd448bd30a/sample2_align2genome.bam
sample3 ->/tmp/Rtmp2P9shN/file397bbd448bd30a/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:54:26 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Nov 16 23:54:27 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp2P9shN/file397bbd448bd30a/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp2P9shN/file397bbd448bd30a/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp2P9shN/file397bbd448bd30a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Nov 16 23:54:45 2025 ----------
2025-11-17T04:54:45.726941Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:54:45.727398Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd448bd30a/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T04:54:45.727420Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:54:45.727473Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:54:45.727546Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:54:45.727564Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T04:54:45.730217Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:54:45.730410Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:54:45.730455Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-11-17T04:54:45.730463Z  INFO oarfish::bulk: number of aligned reads : 98
2025-11-17T04:54:45.730470Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-17T04:54:45.731122Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:54:45.742978Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:54:45.743442Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd448bd30a/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T04:54:45.743464Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:54:45.743472Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:54:45.743560Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:54:45.743573Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T04:54:45.746209Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:54:45.746364Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:54:45.746422Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-11-17T04:54:45.746429Z  INFO oarfish::bulk: number of aligned reads : 97
2025-11-17T04:54:45.746453Z  INFO oarfish::bulk: number of unique alignments : 79
2025-11-17T04:54:45.747102Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:54:45.758904Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:54:45.759420Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd448bd30a/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T04:54:45.759476Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:54:45.759484Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:54:45.759560Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:54:45.759581Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T04:54:45.764217Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:54:45.764424Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-11-17T04:54:45.764491Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-11-17T04:54:45.764498Z  INFO oarfish::bulk: number of aligned reads : 187
2025-11-17T04:54:45.764505Z  INFO oarfish::bulk: number of unique alignments : 140
2025-11-17T04:54:45.765272Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd16a9b699/config_file_3767229.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 @ Sun Nov 16 23:54:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp2P9shN/file397bbd16a9b699/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp2P9shN/file397bbd16a9b699/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp2P9shN/file397bbd16a9b699/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:54:46 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Nov 16 23:54:47 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp2P9shN/file397bbd16a9b699/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmp2P9shN/file397bbd16a9b699/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmp2P9shN/file397bbd16a9b699/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sun Nov 16 23:54:48 2025 ----------
23:54:48 Sun Nov 16 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd57662135/config_file_3767229.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 @ Sun Nov 16 23:54:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp2P9shN/file397bbd57662135/sample1_align2genome.bam
sample2 ->/tmp/Rtmp2P9shN/file397bbd57662135/sample2_align2genome.bam
sample3 ->/tmp/Rtmp2P9shN/file397bbd57662135/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:55:08 2025 -------------
Inputs:  ['/tmp/Rtmp2P9shN/file397bbd16a9b699/sample1_realign2transcript.bam', '/tmp/Rtmp2P9shN/file397bbd16a9b699/sample2_realign2transcript.bam', '/tmp/Rtmp2P9shN/file397bbd16a9b699/sample3_realign2transcript.bam'] /tmp/Rtmp2P9shN/file397bbd16a9b699/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Nov 16 23:55:08 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp2P9shN/file397bbd57662135/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp2P9shN/file397bbd57662135/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp2P9shN/file397bbd57662135/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sun Nov 16 23:55:26 2025 ----------
23:55:26 Sun Nov 16 2025 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)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd41262adf/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Nov 16 23:55:27 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd41262adf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Nov 16 23:55:28 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp2P9shN/file397bbd41262adf/matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd41262adf/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:55:28 2025 -------------
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 @ Sun Nov 16 23:55:38 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd41262adf/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd41262adf/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmp2P9shN/file397bbd41262adf/matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd41262adf/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 @ Sun Nov 16 23:55:38 2025 ----------
2025-11-17T04:55:38.713794Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:55:38.714235Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd41262adf/realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:55:38.714259Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:55:38.714316Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:55:38.714375Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:55:38.714386Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:55:38.720506Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd5465b312/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Nov 16 23:55:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd5465b312/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Nov 16 23:55:39 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd5465b312/matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd5465b312/align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:55:57 2025 -------------
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 @ Sun Nov 16 23:56:07 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd5465b312/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd5465b312/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd5465b312/matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd5465b312/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Nov 16 23:56:26 2025 ----------
2025-11-17T04:56:26.486397Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:56:26.486813Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd5465b312/realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:56:26.486834Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:56:26.486888Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:56:26.486951Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:56:26.486963Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:56:26.493668Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd708b70ea/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Nov 16 23:56:27 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd708b70ea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Nov 16 23:56:27 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp2P9shN/file397bbd708b70ea/matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd708b70ea/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:56:27 2025 -------------
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 @ Sun Nov 16 23:56:37 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd708b70ea/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd708b70ea/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmp2P9shN/file397bbd708b70ea/matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd708b70ea/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 @ Sun Nov 16 23:56:37 2025 ----------
23:56:37 Sun Nov 16 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/Rtmp2P9shN/file397bbd57662135/sample1_realign2transcript.bam', '/tmp/Rtmp2P9shN/file397bbd57662135/sample2_realign2transcript.bam', '/tmp/Rtmp2P9shN/file397bbd57662135/sample3_realign2transcript.bam'] /tmp/Rtmp2P9shN/file397bbd57662135/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd68b9590e/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Nov 16 23:56:38 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd68b9590e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Nov 16 23:56:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd68b9590e/matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd68b9590e/align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:56:58 2025 -------------
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 @ Sun Nov 16 23:57:08 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd68b9590e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd68b9590e/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd68b9590e/matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd68b9590e/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Nov 16 23:57:25 2025 ----------
23:57:25 Sun Nov 16 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd8dd2d62/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Nov 16 23:57:26 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd8dd2d62/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Nov 16 23:57:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp2P9shN/file397bbd8dd2d62/matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd8dd2d62/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:57:27 2025 -------------
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Nov 16 23:57:27 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd8dd2d62/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd8dd2d62/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmp2P9shN/file397bbd8dd2d62/matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd8dd2d62/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 @ Sun Nov 16 23:57:27 2025 ----------
2025-11-17T04:57:27.916590Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:57:27.917038Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd8dd2d62/realign2transcript.bam, contains 10 reference sequences.
2025-11-17T04:57:27.917097Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:57:27.917106Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:57:27.917189Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:57:27.917202Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T04:57:27.926917Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd1740a93d/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Nov 16 23:57:28 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd1740a93d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Nov 16 23:57:28 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd1740a93d/matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd1740a93d/align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:57:46 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Nov 16 23:57:46 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd1740a93d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd1740a93d/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd1740a93d/matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd1740a93d/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Nov 16 23:58:05 2025 ----------
2025-11-17T04:58:05.547681Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:58:05.548134Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd1740a93d/realign2transcript.bam, contains 10 reference sequences.
2025-11-17T04:58:05.548158Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:58:05.548165Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:58:05.548287Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:58:05.548300Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T04:58:05.557926Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd60c3ee07/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Nov 16 23:58:06 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd60c3ee07/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Nov 16 23:58:06 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp2P9shN/file397bbd60c3ee07/matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd60c3ee07/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:58:07 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Nov 16 23:58:07 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd60c3ee07/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd60c3ee07/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmp2P9shN/file397bbd60c3ee07/matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd60c3ee07/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 @ Sun Nov 16 23:58:07 2025 ----------
23:58:07 Sun Nov 16 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd5e2c66cb/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sun Nov 16 23:58:09 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd5e2c66cb/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Sun Nov 16 23:58:09 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd5e2c66cb/matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd5e2c66cb/align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:58:27 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sun Nov 16 23:58:28 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd5e2c66cb/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd5e2c66cb/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd5e2c66cb/matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd5e2c66cb/realign2transcript.bam
-- Running step: transcript_quantification @ Sun Nov 16 23:58:46 2025 ----------
23:58:46 Sun Nov 16 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd3060de2c/config_file_3767229.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 @ Sun Nov 16 23:58:47 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd3060de2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd3060de2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd3060de2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd3060de2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Nov 16 23:58:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sun Nov 16 23:58:50 2025 ----------------
23:58:50 Sun Nov 16 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_align2genome.bam', and
'/tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.94gene_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, 424834.29Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 43.06gene_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, 1309574.12Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.56gene_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, 1239158.59Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.58gene_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, 736049.42Read/s]
-- Running step: isoform_identification @ Sun Nov 16 23:58:52 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 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 @ Sun Nov 16 23:59:15 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq, /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample1.fq.gz, /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample2.fq.gz, /tmp/Rtmp2P9shN/file397bbd3060de2c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sun Nov 16 23:59:15 2025 ----------
2025-11-17T04:59:16.009745Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:59:16.010163Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd3060de2c/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:59:16.010243Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:59:16.010251Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:59:16.010314Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:59:16.010340Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:59:16.016556Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-17T04:59:16.342159Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:59:16.342550Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd3060de2c/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:59:16.342615Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:59:16.342624Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:59:16.342681Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:59:16.342704Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:59:16.662130Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:59:16.662597Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd3060de2c/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:59:16.662619Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:59:16.662672Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:59:16.662741Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:59:16.662754Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:59:16.951374Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:59:16.951813Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd3060de2c/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:59:16.951832Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:59:16.951846Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:59:16.951929Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:59:16.951940Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd2a5c1b76/config_file_3767229.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 @ Sun Nov 16 23:59:17 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd2a5c1b76/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd2a5c1b76/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd2a5c1b76/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd2a5c1b76/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sun Nov 16 23:59:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_align2genome.bam
-- Running step: gene_quantification @ Sun Nov 16 23:59:37 2025 ----------------
23:59:37 Sun Nov 16 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_align2genome.bam', and
'/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_align2genome.bam'
parsing /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.76gene_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, 373185.28Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.09gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1532781.76Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.80gene_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, 1340375.81Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.40gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 710754.42Read/s]
-- Running step: isoform_identification @ Sun Nov 16 23:59:38 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:00:02 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample1.fq.gz, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample2.fq.gz, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:00:20 2025 ----------
2025-11-17T05:00:20.658573Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:00:20.659416Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:00:20.659491Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:00:20.659499Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:00:20.659554Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:00:20.659565Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:00:20.665499Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-17T05:00:21.032246Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:00:21.032606Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:00:21.032673Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:00:21.032681Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:00:21.032735Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:00:21.032745Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:00:21.348016Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:00:21.348439Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:00:21.348458Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:00:21.348466Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:00:21.348595Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:00:21.348606Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:00:21.675217Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:00:21.675703Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd2a5c1b76/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:00:21.675756Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:00:21.675764Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:00:21.675817Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:00:21.675852Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd4e79d2d0/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Nov 17 00:00:22 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd4e79d2d0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd4e79d2d0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd4e79d2d0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd4e79d2d0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Nov 17 00:00:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd4e79d2d0/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/Rtmp2P9shN/file397bbd4e79d2d0/sample1_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd4e79d2d0/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/Rtmp2P9shN/file397bbd4e79d2d0/sample2_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd4e79d2d0/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/Rtmp2P9shN/file397bbd4e79d2d0/sample3_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Nov 17 00:00:24 2025 ----------------
00:00:24 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample1_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample2_align2genome.bam', and
'/tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_align2genome.bam'
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.96gene_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, 374638.61Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.91gene_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, 1410134.48Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.94gene_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, 1337469.39Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 674065.31Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:00:25 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:00:47 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample1.fq.gz, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample2.fq.gz, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample1_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample2_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd4e79d2d0/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/Rtmp2P9shN/file397bbd4e79d2d0/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd4e79d2d0/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/Rtmp2P9shN/file397bbd4e79d2d0/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd4e79d2d0/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/Rtmp2P9shN/file397bbd4e79d2d0/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Nov 17 00:00:48 2025 ----------
00:00:48 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd4e79d2d0/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample1_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample2_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd4e79d2d0/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd3ea9c082/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Nov 17 00:00:50 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd3ea9c082/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd3ea9c082/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd3ea9c082/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd3ea9c082/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Nov 17 00:00:50 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Nov 17 00:01:11 2025 ----------------
00:01:11 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_align2genome.bam', and
'/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 366365.96Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 31.54gene_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, 1302417.09Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.74gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1358611.04Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 686106.13Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:01:12 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:01:34 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq, /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample1.fq.gz, /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample2.fq.gz, /tmp/Rtmp2P9shN/file397bbd3ea9c082/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:01:52 2025 ----------
00:01:52 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd3ea9c082/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd3ea9c082/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd14432722/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Nov 17 00:01:54 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd14432722/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd14432722/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd14432722/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd14432722/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Nov 17 00:01:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp2P9shN/file397bbd14432722/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd14432722/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp2P9shN/file397bbd14432722/sample1_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd14432722/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp2P9shN/file397bbd14432722/sample2_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd14432722/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp2P9shN/file397bbd14432722/sample3_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd14432722/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Nov 17 00:01:57 2025 ----------------
00:01:57 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp2P9shN/file397bbd14432722/sampleA_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd14432722/sample1_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd14432722/sample2_align2genome.bam', and
'/tmp/Rtmp2P9shN/file397bbd14432722/sample3_align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
	Counter({'counted_reads': 89, 'not_enough_coverage': 2})
	Counter({'counted_reads': 92, 'not_enough_coverage': 3})
	Counter({'counted_reads': 168, 'not_enough_coverage': 6, 'unmapped': 2})
parsing /tmp/Rtmp2P9shN/file397bbd14432722/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.56gene_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, 377443.58Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd14432722/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.70gene_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, 1269462.47Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd14432722/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.38gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1292623.27Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd14432722/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.08gene_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, 727066.98Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:01:57 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:01:58 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd14432722/fastq, /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample1.fq.gz, /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample2.fq.gz, /tmp/Rtmp2P9shN/file397bbd14432722/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd14432722/sampleA_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd14432722/sample1_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd14432722/sample2_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd14432722/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd14432722/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd14432722/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd14432722/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd14432722/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp2P9shN/file397bbd14432722/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd14432722/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp2P9shN/file397bbd14432722/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd14432722/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp2P9shN/file397bbd14432722/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd14432722/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp2P9shN/file397bbd14432722/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd14432722/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Nov 17 00:02:00 2025 ----------
2025-11-17T05:02:00.244381Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:00.244970Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd14432722/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:02:00.244994Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:00.245003Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:00.245091Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:00.245106Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:02:00.257267Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-17T05:02:00.912559Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:00.913063Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd14432722/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:02:00.913088Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:00.913097Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:00.913180Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:00.913195Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:02:01.470048Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:01.470612Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd14432722/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:02:01.470635Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:01.470644Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:01.470729Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:01.470743Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:02:02.065765Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:02.066387Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd14432722/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:02:02.066413Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:02.066421Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:02.066511Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:02.066525Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd4c10af24/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Nov 17 00:02:02 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd4c10af24/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd4c10af24/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd4c10af24/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd4c10af24/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Nov 17 00:02:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Nov 17 00:02:23 2025 ----------------
00:02:23 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_align2genome.bam', and
'/tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_align2genome.bam'
parsing /tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.36gene_group/s]
/home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 373118.88Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.95gene_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, 1183761.57Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.30gene_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, 1164696.21Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.73gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 723555.06Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:02:24 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:02:24 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq, /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample1.fq.gz, /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample2.fq.gz, /tmp/Rtmp2P9shN/file397bbd4c10af24/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:02:44 2025 ----------
2025-11-17T05:02:44.803548Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:44.803957Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd4c10af24/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:02:44.803980Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:44.803988Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:44.804069Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:44.804083Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:02:44.815929Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-17T05:02:45.464929Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:45.465345Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd4c10af24/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:02:45.465368Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:45.465377Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:45.465453Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:45.465467Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:02:46.071768Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:46.072177Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd4c10af24/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:02:46.072200Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:46.072208Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:46.072286Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:46.072301Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:02:46.616630Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:46.617035Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp2P9shN/file397bbd4c10af24/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:02:46.617061Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:46.617070Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:46.617151Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:46.617166Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd513b31c7/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Nov 17 00:02:47 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd513b31c7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd513b31c7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd513b31c7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd513b31c7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Nov 17 00:02:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd513b31c7/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/Rtmp2P9shN/file397bbd513b31c7/sample1_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd513b31c7/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/Rtmp2P9shN/file397bbd513b31c7/sample2_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd513b31c7/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/Rtmp2P9shN/file397bbd513b31c7/sample3_matched_reads.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Nov 17 00:02:49 2025 ----------------
00:02:49 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd513b31c7/sample1_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd513b31c7/sample2_align2genome.bam', and
'/tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_align2genome.bam'
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.60gene_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, 435726.57Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.50gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1408240.67Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1227696.99Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.01gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 742512.39Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:02:50 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:02:51 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq, /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample1.fq.gz, /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample2.fq.gz, /tmp/Rtmp2P9shN/file397bbd513b31c7/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd513b31c7/sample1_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd513b31c7/sample2_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd513b31c7/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd513b31c7/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd513b31c7/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/Rtmp2P9shN/file397bbd513b31c7/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd513b31c7/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/Rtmp2P9shN/file397bbd513b31c7/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd513b31c7/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/Rtmp2P9shN/file397bbd513b31c7/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Nov 17 00:02:52 2025 ----------
00:02:52 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd513b31c7/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample1_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd513b31c7/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample2_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd513b31c7/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd513b31c7/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp2P9shN/file397bbd8a62569/config_file_3767229.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Mon Nov 17 00:02:55 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd8a62569/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd8a62569/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample1.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 92
Number of reads with exactly one barcode match: 91
Number of chimera reads: 1
All done!
Reads	Barcodes
4	1
3	9
2	9
1	44
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd8a62569/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample2.fq.gz
Searching for barcodes...
Number of reads processed: 100
Number of reads where at least one barcode was found: 95
Number of reads with exactly one barcode match: 94
Number of chimera reads: 0
All done!
Reads	Barcodes
4	2
3	3
2	16
1	47
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmp2P9shN/file397bbd8a62569/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Mon Nov 17 00:02:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd8a62569/sample1_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd8a62569/sample1_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd8a62569/sample2_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd8a62569/sample2_align2genome.bam
/tmp/Rtmp2P9shN/file397bbd8a62569/sample3_matched_reads.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd8a62569/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Nov 17 00:03:16 2025 ----------------
00:03:16 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd8a62569/sample1_align2genome.bam',
'/tmp/Rtmp2P9shN/file397bbd8a62569/sample2_align2genome.bam', and
'/tmp/Rtmp2P9shN/file397bbd8a62569/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.84gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 337890.63Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.49gene_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, 1234344.91Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.67gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1252330.11Read/s]
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.81gene_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, 729089.14Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:03:17 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:03:17 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd8a62569/fastq, /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample1.fq.gz, /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample2.fq.gz, /tmp/Rtmp2P9shN/file397bbd8a62569/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd8a62569/sample1_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd8a62569/sample2_matched_reads.fastq.gz, /tmp/Rtmp2P9shN/file397bbd8a62569/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd8a62569/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd8a62569/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp2P9shN/file397bbd8a62569/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd8a62569/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd8a62569/sample1_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd8a62569/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd8a62569/sample2_realign2transcript.bam
/tmp/Rtmp2P9shN/file397bbd8a62569/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp2P9shN/file397bbd8a62569/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:03:37 2025 ----------
00:03:37 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd8a62569/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample1_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd8a62569/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample2_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd8a62569/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample3_realign2transcript.bam...
parsing /tmp/Rtmp2P9shN/file397bbd8a62569/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp2P9shN/file397bbd8a62569/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
> 
> proc.time()
   user  system elapsed 
727.633  46.405 760.603