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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 (2025-06-13)
* 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 ‘2.3.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     23.397  1.094  24.491
find_variants                22.375  1.473  23.241
blaze                         4.541 13.848  11.280
sc_long_multisample_pipeline  8.162  5.560   7.754
bulk_long_pipeline            2.410 10.421   2.505
MultiSampleSCPipeline        10.404  0.643  11.451
sc_plot_genotype             10.574  0.257   9.681
sc_DTU_analysis               7.075  1.730   6.945
plot_isoform_heatmap          7.348  0.554   7.902
create_sce_from_dir           3.563  1.995   3.680
* 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-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
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* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.3.5’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R version 4.5.1 (2025-06-13) -- "Great Square Root"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a516124834/config_file_1778341.json 
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a516124834/config_file_1778341.json 
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a516124834/config_file_1778341.json 
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a5651e1aa5/config_file_1778341.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/RtmpqM1eJC/file1b22a54aa1ecc4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpqM1eJC/file1b22a516296a56/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpqM1eJC/file1b22a516296a56/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a565b2a24/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a565b2a24/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a565b2a24/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a565b2a24/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/RtmpqM1eJC/file1b22a5af748d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a51377154f/config_file_1778341.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 @ Thu Sep  4 23:54:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpqM1eJC/file1b22a51377154f/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpqM1eJC/file1b22a51377154f/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpqM1eJC/file1b22a51377154f/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Sep  4 23:54:27 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:54:33] 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.

[23:54:33] 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.

[23:54:34] 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.

[23:54:34] 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.

[23:54:35] 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.

[23:54:36] 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 @ Thu Sep  4 23:54:53 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpqM1eJC/file1b22a51377154f/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpqM1eJC/file1b22a51377154f/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpqM1eJC/file1b22a51377154f/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Thu Sep  4 23:54:53 2025 ----------
2025-09-05T03:54:53.654412Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:54:53.654844Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a51377154f/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T03:54:53.654857Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:54:53.654861Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:54:53.654935Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:54:53.654942Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T03:54:53.656499Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:54:53.656635Z  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    │
╰─────────────────────────────────┴───────╯

2025-09-05T03:54:53.656657Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-09-05T03:54:53.656673Z  INFO oarfish::bulk: number of aligned reads : 96
2025-09-05T03:54:53.656675Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-05T03:54:53.657326Z  INFO oarfish: oarfish completed successfully.
2025-09-05T03:54:53.664787Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:54:53.665162Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a51377154f/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T03:54:53.665171Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:54:53.665174Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:54:53.665231Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:54:53.665237Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T03:54:53.666880Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:54:53.667025Z  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    │
╰─────────────────────────────────┴───────╯

2025-09-05T03:54:53.667051Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-09-05T03:54:53.667054Z  INFO oarfish::bulk: number of aligned reads : 95
2025-09-05T03:54:53.667057Z  INFO oarfish::bulk: number of unique alignments : 82
2025-09-05T03:54:53.667663Z  INFO oarfish: oarfish completed successfully.
2025-09-05T03:54:53.674759Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:54:53.675124Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a51377154f/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T03:54:53.675132Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:54:53.675135Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:54:53.675189Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:54:53.675194Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T03:54:53.677777Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-09-05T03:54:53.677937Z  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   │
╰─────────────────────────────────┴───────╯

2025-09-05T03:54:53.677975Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-09-05T03:54:53.677978Z  INFO oarfish::bulk: number of aligned reads : 179
2025-09-05T03:54:53.677980Z  INFO oarfish::bulk: number of unique alignments : 143
2025-09-05T03:54:53.678634Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a55c36f2e5/config_file_1778341.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 @ Thu Sep  4 23:54:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample1_align2genome.bam
sample2 ->/tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample2_align2genome.bam
sample3 ->/tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu Sep  4 23:55:15 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 @ Thu Sep  4 23:55:37 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Sep  4 23:55:57 2025 ----------
2025-09-05T03:55:57.392538Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:55:57.393003Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T03:55:57.393029Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:55:57.393032Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:55:57.393094Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:55:57.393099Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T03:55:57.394634Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:55:57.394766Z  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    │
╰─────────────────────────────────┴───────╯

2025-09-05T03:55:57.394794Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-09-05T03:55:57.394797Z  INFO oarfish::bulk: number of aligned reads : 96
2025-09-05T03:55:57.394799Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-05T03:55:57.395388Z  INFO oarfish: oarfish completed successfully.
2025-09-05T03:55:57.406168Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:55:57.406527Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T03:55:57.406534Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:55:57.406537Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:55:57.406589Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:55:57.406595Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T03:55:57.408125Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:55:57.408241Z  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    │
╰─────────────────────────────────┴───────╯

2025-09-05T03:55:57.408265Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-09-05T03:55:57.408278Z  INFO oarfish::bulk: number of aligned reads : 95
2025-09-05T03:55:57.408280Z  INFO oarfish::bulk: number of unique alignments : 82
2025-09-05T03:55:57.408850Z  INFO oarfish: oarfish completed successfully.
2025-09-05T03:55:57.419469Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:55:57.419830Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55c36f2e5/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T03:55:57.419841Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:55:57.419843Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:55:57.419897Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:55:57.419902Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T03:55:57.422589Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-09-05T03:55:57.422756Z  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   │
╰─────────────────────────────────┴───────╯

2025-09-05T03:55:57.422786Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-09-05T03:55:57.422789Z  INFO oarfish::bulk: number of aligned reads : 179
2025-09-05T03:55:57.422791Z  INFO oarfish::bulk: number of unique alignments : 143
2025-09-05T03:55:57.423459Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a55d7e7931/config_file_1778341.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 @ Thu Sep  4 23:55:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpqM1eJC/file1b22a55d7e7931/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpqM1eJC/file1b22a55d7e7931/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpqM1eJC/file1b22a55d7e7931/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Sep  4 23:55:58 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 @ Thu Sep  4 23:56:18 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpqM1eJC/file1b22a55d7e7931/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpqM1eJC/file1b22a55d7e7931/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpqM1eJC/file1b22a55d7e7931/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Thu Sep  4 23:56:18 2025 ----------
23:56:18 Thu Sep 04 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/RtmpqM1eJC/file1b22a57e6e6cc0/config_file_1778341.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 @ Thu Sep  4 23:56:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample1_align2genome.bam
sample2 ->/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample2_align2genome.bam
sample3 ->/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu Sep  4 23:56:39 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 @ Thu Sep  4 23:57:00 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Sep  4 23:57:19 2025 ----------
23:57:19 Thu Sep 04 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpqM1eJC/file1b22a55d7e7931/sample1_realign2transcript.bam', '/tmp/RtmpqM1eJC/file1b22a55d7e7931/sample2_realign2transcript.bam', '/tmp/RtmpqM1eJC/file1b22a55d7e7931/sample3_realign2transcript.bam'] /tmp/RtmpqM1eJC/file1b22a55d7e7931/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/RtmpqM1eJC/file1b22a515bb8618/config_file_1778341.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 @ Thu Sep  4 23:57:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpqM1eJC/file1b22a515bb8618/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpqM1eJC/file1b22a515bb8618/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpqM1eJC/file1b22a515bb8618/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Sep  4 23:57:21 2025 -------------
Inputs:  ['/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample1_realign2transcript.bam', '/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample2_realign2transcript.bam', '/tmp/RtmpqM1eJC/file1b22a57e6e6cc0/sample3_realign2transcript.bam'] /tmp/RtmpqM1eJC/file1b22a57e6e6cc0/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 @ Thu Sep  4 23:57:21 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpqM1eJC/file1b22a515bb8618/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpqM1eJC/file1b22a515bb8618/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpqM1eJC/file1b22a515bb8618/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Thu Sep  4 23:57:22 2025 ----------
2025-09-05T03:57:22.526932Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:57:22.527319Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a515bb8618/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-09-05T03:57:22.527330Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:57:22.527334Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:57:22.527404Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:57:22.527412Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-05T03:57:22.530026Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:57:22.530180Z  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    │
╰─────────────────────────────────┴───────╯

2025-09-05T03:57:22.530203Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-09-05T03:57:22.530206Z  INFO oarfish::bulk: number of aligned reads : 98
2025-09-05T03:57:22.530208Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-05T03:57:22.530866Z  INFO oarfish: oarfish completed successfully.
2025-09-05T03:57:22.538456Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:57:22.538849Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a515bb8618/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-09-05T03:57:22.538861Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:57:22.538864Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:57:22.538940Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:57:22.538947Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-05T03:57:22.541575Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:57:22.541742Z  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    │
╰─────────────────────────────────┴───────╯

2025-09-05T03:57:22.541771Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-09-05T03:57:22.541774Z  INFO oarfish::bulk: number of aligned reads : 97
2025-09-05T03:57:22.541776Z  INFO oarfish::bulk: number of unique alignments : 79
2025-09-05T03:57:22.542409Z  INFO oarfish: oarfish completed successfully.
2025-09-05T03:57:22.550027Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:57:22.550374Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a515bb8618/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-09-05T03:57:22.550383Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:57:22.550386Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:57:22.550456Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:57:22.550464Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-05T03:57:22.554698Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:57:22.554923Z  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   │
╰─────────────────────────────────┴───────╯

2025-09-05T03:57:22.554959Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-09-05T03:57:22.554962Z  INFO oarfish::bulk: number of aligned reads : 187
2025-09-05T03:57:22.554965Z  INFO oarfish::bulk: number of unique alignments : 140
2025-09-05T03:57:22.555702Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a57743ec81/config_file_1778341.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 @ Thu Sep  4 23:57:22 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpqM1eJC/file1b22a57743ec81/sample1_align2genome.bam
sample2 ->/tmp/RtmpqM1eJC/file1b22a57743ec81/sample2_align2genome.bam
sample3 ->/tmp/RtmpqM1eJC/file1b22a57743ec81/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu Sep  4 23:57:41 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Sep  4 23:57:42 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpqM1eJC/file1b22a57743ec81/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpqM1eJC/file1b22a57743ec81/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpqM1eJC/file1b22a57743ec81/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Sep  4 23:58:01 2025 ----------
2025-09-05T03:58:01.761448Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:58:01.761943Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a57743ec81/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-09-05T03:58:01.761957Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:58:01.761973Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:58:01.762049Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:58:01.762056Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-05T03:58:01.764758Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:58:01.764917Z  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    │
╰─────────────────────────────────┴───────╯

2025-09-05T03:58:01.764941Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-09-05T03:58:01.764955Z  INFO oarfish::bulk: number of aligned reads : 98
2025-09-05T03:58:01.764957Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-05T03:58:01.765602Z  INFO oarfish: oarfish completed successfully.
2025-09-05T03:58:01.777178Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:58:01.777575Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a57743ec81/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-09-05T03:58:01.777584Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:58:01.777587Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:58:01.777660Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:58:01.777668Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-05T03:58:01.780458Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:58:01.780602Z  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    │
╰─────────────────────────────────┴───────╯

2025-09-05T03:58:01.780628Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-09-05T03:58:01.780631Z  INFO oarfish::bulk: number of aligned reads : 97
2025-09-05T03:58:01.780645Z  INFO oarfish::bulk: number of unique alignments : 79
2025-09-05T03:58:01.781276Z  INFO oarfish: oarfish completed successfully.
2025-09-05T03:58:01.791018Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:58:01.791378Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a57743ec81/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-09-05T03:58:01.791388Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:58:01.791391Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:58:01.791461Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:58:01.791468Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-05T03:58:01.795776Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-05T03:58:01.795951Z  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   │
╰─────────────────────────────────┴───────╯

2025-09-05T03:58:01.795979Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-09-05T03:58:01.795982Z  INFO oarfish::bulk: number of aligned reads : 187
2025-09-05T03:58:01.795985Z  INFO oarfish::bulk: number of unique alignments : 140
2025-09-05T03:58:01.796728Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a5205610cd/config_file_1778341.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 @ Thu Sep  4 23:58:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpqM1eJC/file1b22a5205610cd/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpqM1eJC/file1b22a5205610cd/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpqM1eJC/file1b22a5205610cd/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Sep  4 23:58:02 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Thu Sep  4 23:58:03 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpqM1eJC/file1b22a5205610cd/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpqM1eJC/file1b22a5205610cd/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpqM1eJC/file1b22a5205610cd/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Thu Sep  4 23:58:03 2025 ----------
23:58:03 Thu Sep 04 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/RtmpqM1eJC/file1b22a56a0df2a0/config_file_1778341.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 @ Thu Sep  4 23:58:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample1_align2genome.bam
sample2 ->/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample2_align2genome.bam
sample3 ->/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample3_align2genome.bam
-- Running step: isoform_identification @ Thu Sep  4 23:58:25 2025 -------------
Inputs:  ['/tmp/RtmpqM1eJC/file1b22a5205610cd/sample1_realign2transcript.bam', '/tmp/RtmpqM1eJC/file1b22a5205610cd/sample2_realign2transcript.bam', '/tmp/RtmpqM1eJC/file1b22a5205610cd/sample3_realign2transcript.bam'] /tmp/RtmpqM1eJC/file1b22a5205610cd/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 @ Thu Sep  4 23:58:26 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Thu Sep  4 23:58:44 2025 ----------
23:58:44 Thu Sep 04 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/RtmpqM1eJC/file1b22a5429bd761/config_file_1778341.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 @ Thu Sep  4 23:58:45 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/RtmpqM1eJC/file1b22a5429bd761/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Thu Sep  4 23:58:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqM1eJC/file1b22a5429bd761/matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5429bd761/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Thu Sep  4 23:58:46 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 @ Thu Sep  4 23:58:56 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a5429bd761/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a5429bd761/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpqM1eJC/file1b22a5429bd761/matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5429bd761/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 @ Thu Sep  4 23:58:56 2025 ----------
2025-09-05T03:58:56.835954Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:58:56.836640Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a5429bd761/realign2transcript.bam, contains 5 reference sequences.
2025-09-05T03:58:56.836653Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:58:56.836656Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:58:56.836729Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:58:56.836737Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T03:58:56.843143Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a57ad3fa70/config_file_1778341.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 @ Thu Sep  4 23:58:57 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/RtmpqM1eJC/file1b22a57ad3fa70/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Thu Sep  4 23:58:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a57ad3fa70/matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57ad3fa70/align2genome.bam
-- Running step: isoform_identification @ Thu Sep  4 23:59:16 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 @ Thu Sep  4 23:59:28 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a57ad3fa70/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a57ad3fa70/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a57ad3fa70/matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57ad3fa70/realign2transcript.bam
-- Running step: transcript_quantification @ Thu Sep  4 23:59:47 2025 ----------
2025-09-05T03:59:48.010688Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T03:59:48.011148Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a57ad3fa70/realign2transcript.bam, contains 5 reference sequences.
2025-09-05T03:59:48.011161Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T03:59:48.011165Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T03:59:48.011224Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T03:59:48.011230Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T03:59:48.018544Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a53e3ac1e9/config_file_1778341.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 @ Thu Sep  4 23:59:48 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/RtmpqM1eJC/file1b22a53e3ac1e9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Thu Sep  4 23:59:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqM1eJC/file1b22a53e3ac1e9/matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53e3ac1e9/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 @ Thu Sep  4 23:59:49 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 @ Thu Sep  4 23:59:59 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a53e3ac1e9/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a53e3ac1e9/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpqM1eJC/file1b22a53e3ac1e9/matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53e3ac1e9/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Sep  5 00:00:00 2025 ----------
00:00:00 Fri Sep 05 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample1_realign2transcript.bam', '/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample2_realign2transcript.bam', '/tmp/RtmpqM1eJC/file1b22a56a0df2a0/sample3_realign2transcript.bam'] /tmp/RtmpqM1eJC/file1b22a56a0df2a0/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/RtmpqM1eJC/file1b22a53f203396/config_file_1778341.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 @ Fri Sep  5 00:00:00 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/RtmpqM1eJC/file1b22a53f203396/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Sep  5 00:00:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a53f203396/matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a53f203396/align2genome.bam
-- Running step: isoform_identification @ Fri Sep  5 00:00:20 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 @ Fri Sep  5 00:00:30 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a53f203396/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a53f203396/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a53f203396/matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a53f203396/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Sep  5 00:00:48 2025 ----------
00:00:48 Fri Sep 05 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/RtmpqM1eJC/file1b22a57f2af53c/config_file_1778341.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 @ Fri Sep  5 00:00:49 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/RtmpqM1eJC/file1b22a57f2af53c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Sep  5 00:00:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqM1eJC/file1b22a57f2af53c/matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a57f2af53c/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Sep  5 00:00:50 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 @ Fri Sep  5 00:00:50 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a57f2af53c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a57f2af53c/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpqM1eJC/file1b22a57f2af53c/matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a57f2af53c/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Fri Sep  5 00:00:50 2025 ----------
2025-09-05T04:00:50.860603Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:00:50.861032Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a57f2af53c/realign2transcript.bam, contains 10 reference sequences.
2025-09-05T04:00:50.861042Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:00:50.861045Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:00:50.861114Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:00:50.861121Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-05T04:00:50.870765Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a578ff3a8e/config_file_1778341.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 @ Fri Sep  5 00:00:51 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpqM1eJC/file1b22a578ff3a8e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Sep  5 00:00:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a578ff3a8e/matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a578ff3a8e/align2genome.bam
-- Running step: isoform_identification @ Fri Sep  5 00:01:10 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Sep  5 00:01:10 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a578ff3a8e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a578ff3a8e/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a578ff3a8e/matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a578ff3a8e/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Sep  5 00:01:29 2025 ----------
2025-09-05T04:01:29.919739Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:01:29.920152Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a578ff3a8e/realign2transcript.bam, contains 10 reference sequences.
2025-09-05T04:01:29.920163Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:01:29.920166Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:01:29.920251Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:01:29.920259Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-05T04:01:29.931024Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a54a06bc75/config_file_1778341.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 @ Fri Sep  5 00:01:30 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/RtmpqM1eJC/file1b22a54a06bc75/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Sep  5 00:01:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqM1eJC/file1b22a54a06bc75/matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a54a06bc75/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 @ Fri Sep  5 00:01:31 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Sep  5 00:01:31 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a54a06bc75/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a54a06bc75/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpqM1eJC/file1b22a54a06bc75/matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a54a06bc75/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Fri Sep  5 00:01:31 2025 ----------
00:01:31 Fri Sep 05 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a520f088f2/config_file_1778341.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 @ Fri Sep  5 00:01:33 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/RtmpqM1eJC/file1b22a520f088f2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Fri Sep  5 00:01:33 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a520f088f2/matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a520f088f2/align2genome.bam
-- Running step: isoform_identification @ Fri Sep  5 00:01:52 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Sep  5 00:01:53 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a520f088f2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a520f088f2/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a520f088f2/matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a520f088f2/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Sep  5 00:02:10 2025 ----------
00:02:10 Fri Sep 05 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a55f26fe7b/config_file_1778341.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Sep  5 00:02:12 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/RtmpqM1eJC/file1b22a55f26fe7b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a55f26fe7b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a55f26fe7b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a55f26fe7b/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/RtmpqM1eJC/file1b22a55f26fe7b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a55f26fe7b/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/RtmpqM1eJC/file1b22a55f26fe7b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a55f26fe7b/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/RtmpqM1eJC/file1b22a55f26fe7b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a55f26fe7b/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 @ Fri Sep  5 00:02:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Sep  5 00:02:14 2025 ----------------
00:02:14 Fri Sep 05 2025 quantify genes 
Using BAM(s): '/tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_align2genome.bam', and
'/tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 428707.63Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 42.45gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1409944.87Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1287544.20Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 757805.88Read/s]
-- Running step: isoform_identification @ Fri Sep  5 00:02:16 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Sep  5 00:02:41 2025 -------------------
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a55f26fe7b/fastq, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/fastq/sample1.fq.gz, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/fastq/sample2.fq.gz, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Sep  5 00:02:42 2025 ----------
2025-09-05T04:02:42.635971Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:02:42.636316Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T04:02:42.636335Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:02:42.636338Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:02:42.636400Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:02:42.636406Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T04:02:42.642451Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-05T04:02:42.949209Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:02:42.949587Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T04:02:42.949595Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:02:42.949599Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:02:42.949660Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:02:42.949666Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T04:02:43.233162Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:02:43.233548Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T04:02:43.233556Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:02:43.233558Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:02:43.233610Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:02:43.233615Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T04:02:43.513419Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:02:43.513800Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55f26fe7b/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T04:02:43.513812Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:02:43.513815Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:02:43.513873Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:02:43.513879Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a5109d49f4/config_file_1778341.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Sep  5 00:02:44 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/RtmpqM1eJC/file1b22a5109d49f4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a5109d49f4/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a5109d49f4/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a5109d49f4/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/RtmpqM1eJC/file1b22a5109d49f4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a5109d49f4/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/RtmpqM1eJC/file1b22a5109d49f4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a5109d49f4/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/RtmpqM1eJC/file1b22a5109d49f4/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a5109d49f4/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 @ Fri Sep  5 00:02:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Sep  5 00:03:04 2025 ----------------
00:03:04 Fri Sep 05 2025 quantify genes 
Using BAM(s): '/tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_align2genome.bam', and
'/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_align2genome.bam'
parsing /tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.85gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 378670.33Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1403904.14Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.92gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1299028.74Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.29gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 749893.44Read/s]
-- Running step: isoform_identification @ Fri Sep  5 00:03:05 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Sep  5 00:03:30 2025 -------------------
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a5109d49f4/fastq, /tmp/RtmpqM1eJC/file1b22a5109d49f4/fastq/sample1.fq.gz, /tmp/RtmpqM1eJC/file1b22a5109d49f4/fastq/sample2.fq.gz, /tmp/RtmpqM1eJC/file1b22a5109d49f4/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Sep  5 00:03:49 2025 ----------
2025-09-05T04:03:49.520257Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:03:49.520656Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a5109d49f4/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T04:03:49.520667Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:03:49.520671Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:03:49.520737Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:03:49.520745Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T04:03:49.526471Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-05T04:03:49.894995Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:03:49.895388Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T04:03:49.895398Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:03:49.895402Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:03:49.895459Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:03:49.895465Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T04:03:50.209811Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:03:50.210147Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T04:03:50.210157Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:03:50.210160Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:03:50.210211Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:03:50.210217Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-05T04:03:50.536603Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:03:50.536975Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a5109d49f4/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-05T04:03:50.536986Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:03:50.536989Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:03:50.537044Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:03:50.537062Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a5699091e1/config_file_1778341.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Sep  5 00:03:51 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpqM1eJC/file1b22a5699091e1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a5699091e1/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a5699091e1/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a5699091e1/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 @ Fri Sep  5 00:03:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/sample1_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/sample2_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/sample3_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Sep  5 00:03:53 2025 ----------------
00:03:53 Fri Sep 05 2025 quantify genes 
Using BAM(s): '/tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a5699091e1/sample1_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a5699091e1/sample2_align2genome.bam', and
'/tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_align2genome.bam'
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 407198.17Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.14gene_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, 1399967.96Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.40gene_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, 1306636.76Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.06gene_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, 757258.61Read/s]
-- Running step: isoform_identification @ Fri Sep  5 00:03:54 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Sep  5 00:04:21 2025 -------------------
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a5699091e1/fastq, /tmp/RtmpqM1eJC/file1b22a5699091e1/fastq/sample1.fq.gz, /tmp/RtmpqM1eJC/file1b22a5699091e1/fastq/sample2.fq.gz, /tmp/RtmpqM1eJC/file1b22a5699091e1/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5699091e1/sample1_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5699091e1/sample2_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5699091e1/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5699091e1/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5699091e1/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/RtmpqM1eJC/file1b22a5699091e1/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Sep  5 00:04:22 2025 ----------
00:04:22 Fri Sep 05 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a5699091e1/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample1_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a5699091e1/sample1_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample2_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a5699091e1/sample2_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a5699091e1/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a57779f4b6/config_file_1778341.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Sep  5 00:04:24 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/RtmpqM1eJC/file1b22a57779f4b6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a57779f4b6/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a57779f4b6/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a57779f4b6/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/RtmpqM1eJC/file1b22a57779f4b6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a57779f4b6/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/RtmpqM1eJC/file1b22a57779f4b6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a57779f4b6/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/RtmpqM1eJC/file1b22a57779f4b6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a57779f4b6/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 @ Fri Sep  5 00:04:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Sep  5 00:04:45 2025 ----------------
00:04:45 Fri Sep 05 2025 quantify genes 
Using BAM(s): '/tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_align2genome.bam', and
'/tmp/RtmpqM1eJC/file1b22a57779f4b6/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/RtmpqM1eJC/file1b22a57779f4b6/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.27gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 342325.10Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.76gene_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, 1382798.36Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.51gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1280000.00Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.00gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 734039.90Read/s]
-- Running step: isoform_identification @ Fri Sep  5 00:04:46 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%
  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
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  |====================================================                  |  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 @ Fri Sep  5 00:05:13 2025 -------------------
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a57779f4b6/fastq, /tmp/RtmpqM1eJC/file1b22a57779f4b6/fastq/sample1.fq.gz, /tmp/RtmpqM1eJC/file1b22a57779f4b6/fastq/sample2.fq.gz, /tmp/RtmpqM1eJC/file1b22a57779f4b6/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Sep  5 00:05:34 2025 ----------
00:05:34 Fri Sep 05 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a57779f4b6/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample1_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample2_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a57779f4b6/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a534b8eb86/config_file_1778341.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Sep  5 00:05:36 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/RtmpqM1eJC/file1b22a534b8eb86/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a534b8eb86/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a534b8eb86/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a534b8eb86/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/RtmpqM1eJC/file1b22a534b8eb86/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a534b8eb86/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/RtmpqM1eJC/file1b22a534b8eb86/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a534b8eb86/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/RtmpqM1eJC/file1b22a534b8eb86/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a534b8eb86/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 @ Fri Sep  5 00:05:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqM1eJC/file1b22a534b8eb86/sampleA_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a534b8eb86/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample3_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Sep  5 00:05:38 2025 ----------------
00:05:38 Fri Sep 05 2025 quantify genes 
Using BAM(s): '/tmp/RtmpqM1eJC/file1b22a534b8eb86/sampleA_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_align2genome.bam', and
'/tmp/RtmpqM1eJC/file1b22a534b8eb86/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/RtmpqM1eJC/file1b22a534b8eb86/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 404371.60Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1334066.16Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.58gene_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, 1330342.55Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 721017.67Read/s]
-- Running step: isoform_identification @ Fri Sep  5 00:05:39 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Sep  5 00:05:40 2025 -------------------
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a534b8eb86/fastq, /tmp/RtmpqM1eJC/file1b22a534b8eb86/fastq/sample1.fq.gz, /tmp/RtmpqM1eJC/file1b22a534b8eb86/fastq/sample2.fq.gz, /tmp/RtmpqM1eJC/file1b22a534b8eb86/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a534b8eb86/sampleA_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a534b8eb86/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqM1eJC/file1b22a534b8eb86/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a534b8eb86/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Fri Sep  5 00:05:42 2025 ----------
2025-09-05T04:05:42.045213Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:05:42.045591Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a534b8eb86/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-09-05T04:05:42.045600Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:05:42.045603Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:05:42.045682Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:05:42.045690Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-05T04:05:42.057501Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-05T04:05:42.627144Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:05:42.627530Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-09-05T04:05:42.627540Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:05:42.627544Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:05:42.627630Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:05:42.627639Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-05T04:05:43.178357Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:05:43.178755Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-09-05T04:05:43.178766Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:05:43.178769Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:05:43.178858Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:05:43.178866Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-05T04:05:43.780599Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:05:43.781154Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a534b8eb86/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-09-05T04:05:43.781167Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:05:43.781171Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:05:43.781270Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:05:43.781279Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/config_file_1778341.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Sep  5 00:05:44 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/RtmpqM1eJC/file1b22a55ebdfdb1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/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/RtmpqM1eJC/file1b22a55ebdfdb1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/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/RtmpqM1eJC/file1b22a55ebdfdb1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/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/RtmpqM1eJC/file1b22a55ebdfdb1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/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 @ Fri Sep  5 00:05:45 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Sep  5 00:06:05 2025 ----------------
00:06:05 Fri Sep 05 2025 quantify genes 
Using BAM(s): '/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_align2genome.bam', and
'/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_align2genome.bam'
parsing /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 17.80gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 358181.38Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.10gene_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, 1282818.69Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1293580.06Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.72gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 694191.33Read/s]
-- Running step: isoform_identification @ Fri Sep  5 00:06:06 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Sep  5 00:06:07 2025 -------------------
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/fastq, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/fastq/sample1.fq.gz, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/fastq/sample2.fq.gz, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Sep  5 00:06:27 2025 ----------
2025-09-05T04:06:27.753216Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:06:27.753790Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-09-05T04:06:27.753801Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:06:27.753804Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:06:27.753880Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:06:27.753887Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-05T04:06:27.765815Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-05T04:06:28.385783Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:06:28.386204Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-09-05T04:06:28.386214Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:06:28.386218Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:06:28.386319Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:06:28.386328Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-05T04:06:29.015510Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:06:29.015926Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-09-05T04:06:29.015939Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:06:29.015942Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:06:29.016025Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:06:29.016033Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-05T04:06:29.565827Z  INFO oarfish: setting user-provided filter parameters.
2025-09-05T04:06:29.566235Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpqM1eJC/file1b22a55ebdfdb1/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-09-05T04:06:29.566243Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-05T04:06:29.566246Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-05T04:06:29.566322Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-05T04:06:29.566330Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a53c2b2c78/config_file_1778341.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Sep  5 00:06:30 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/RtmpqM1eJC/file1b22a53c2b2c78/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a53c2b2c78/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a53c2b2c78/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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 @ Fri Sep  5 00:06:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/sample1_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/sample2_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/sample3_matched_reads.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Fri Sep  5 00:06:32 2025 ----------------
00:06:32 Fri Sep 05 2025 quantify genes 
Using BAM(s): '/tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample1_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample2_align2genome.bam', and
'/tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_align2genome.bam'
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.09gene_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, 400785.84Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  8.27gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  8.26gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1359139.34Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.22gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1309083.65Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.00gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 736618.19Read/s]
-- Running step: isoform_identification @ Fri Sep  5 00:06:33 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Sep  5 00:06:33 2025 -------------------
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a53c2b2c78/fastq, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/fastq/sample1.fq.gz, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/fastq/sample2.fq.gz, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample1_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample2_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53c2b2c78/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/RtmpqM1eJC/file1b22a53c2b2c78/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Sep  5 00:06:35 2025 ----------
00:06:35 Fri Sep 05 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a53c2b2c78/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample1_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample1_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample2_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample2_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a53c2b2c78/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpqM1eJC/file1b22a54f244685/config_file_1778341.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Fri Sep  5 00:06:38 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpqM1eJC/file1b22a54f244685/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a54f244685/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a54f244685/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpqM1eJC/file1b22a54f244685/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/RtmpqM1eJC/file1b22a54f244685/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a54f244685/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/RtmpqM1eJC/file1b22a54f244685/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a54f244685/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/RtmpqM1eJC/file1b22a54f244685/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpqM1eJC/file1b22a54f244685/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 @ Fri Sep  5 00:06:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a54f244685/sample1_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a54f244685/sample1_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a54f244685/sample2_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a54f244685/sample2_align2genome.bam
/tmp/RtmpqM1eJC/file1b22a54f244685/sample3_matched_reads.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a54f244685/sample3_align2genome.bam
-- Running step: gene_quantification @ Fri Sep  5 00:06:58 2025 ----------------
00:06:58 Fri Sep 05 2025 quantify genes 
Using BAM(s): '/tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a54f244685/sample1_align2genome.bam',
'/tmp/RtmpqM1eJC/file1b22a54f244685/sample2_align2genome.bam', and
'/tmp/RtmpqM1eJC/file1b22a54f244685/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/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, 438459.54Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.78gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1294058.99Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.59gene_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, 1336957.80Read/s]
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.33gene_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, 790900.59Read/s]
-- Running step: isoform_identification @ Fri Sep  5 00:06:59 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Sep  5 00:06:59 2025 -------------------
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a54f244685/fastq, /tmp/RtmpqM1eJC/file1b22a54f244685/fastq/sample1.fq.gz, /tmp/RtmpqM1eJC/file1b22a54f244685/fastq/sample2.fq.gz, /tmp/RtmpqM1eJC/file1b22a54f244685/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a54f244685/sample1_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a54f244685/sample2_matched_reads.fastq.gz, /tmp/RtmpqM1eJC/file1b22a54f244685/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a54f244685/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a54f244685/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpqM1eJC/file1b22a54f244685/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a54f244685/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a54f244685/sample1_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a54f244685/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a54f244685/sample2_realign2transcript.bam
/tmp/RtmpqM1eJC/file1b22a54f244685/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpqM1eJC/file1b22a54f244685/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Sep  5 00:07:19 2025 ----------
00:07:19 Fri Sep 05 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a54f244685/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample1_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a54f244685/sample1_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample2_realign2transcript.bam...
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a54f244685/sample2_realign2transcript.bamdone
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample3_realign2transcript.bam...
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
parsing /tmp/RtmpqM1eJC/file1b22a54f244685/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpqM1eJC/file1b22a54f244685/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': 176})
> 
> proc.time()
    user   system  elapsed 
1703.548   47.356  794.827