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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.4.1.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 Patched (2025-08-23 r88802)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.4.1’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     25.827  1.526  27.351
blaze                         4.598 18.793  13.146
find_variants                21.770  0.066  21.235
sc_long_multisample_pipeline  8.033  5.853   7.972
bulk_long_pipeline            2.364 11.322   2.530
sc_plot_genotype             11.116  0.248  10.224
MultiSampleSCPipeline        10.202  0.635  11.169
sc_DTU_analysis               7.477  2.447   7.563
plot_isoform_heatmap          7.302  0.664   7.964
create_sce_from_dir           3.601  3.031   3.906
* 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 ‘2.4.1’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R version 4.5.1 Patched (2025-08-23 r88802) -- "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/Rtmpv8qU1Q/file1d51b624d88da5/config_file_1921462.json 
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b624d88da5/config_file_1921462.json 
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b624d88da5/config_file_1921462.json 
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b675a57515/config_file_1921462.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/Rtmpv8qU1Q/file1d51b611c84e9c/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/Rtmpv8qU1Q/file1d51b6378e6afe/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/Rtmpv8qU1Q/file1d51b6378e6afe/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6491101c4/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6491101c4/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6491101c4/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6491101c4/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/Rtmpv8qU1Q/file1d51b64949c4d0/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/Rtmpv8qU1Q/file1d51b63bb8d303/config_file_1921462.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Nov 10 23:58:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Nov 10 23:58:05 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:58:13] 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.

[23:58:13] 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.

[23:58:13] 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.

[23:58:13] 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.

[23:58:14] 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.

[23:58:14] 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 @ Mon Nov 10 23:58:30 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Nov 10 23:58:30 2025 ----------
2025-11-11T04:58:30.951398Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T04:58:30.951920Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T04:58:30.951933Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T04:58:30.951937Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T04:58:30.952002Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T04:58:30.952009Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T04:58:30.953538Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T04:58:30.953665Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-11-11T04:58:30.953687Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-11-11T04:58:30.953701Z  INFO oarfish::bulk: number of aligned reads : 96
2025-11-11T04:58:30.953703Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-11T04:58:30.954348Z  INFO oarfish: oarfish completed successfully.
2025-11-11T04:58:30.961790Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T04:58:30.962178Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T04:58:30.962190Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T04:58:30.962194Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T04:58:30.962248Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T04:58:30.962254Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T04:58:30.963750Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T04:58:30.963886Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-11-11T04:58:30.963913Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-11-11T04:58:30.963917Z  INFO oarfish::bulk: number of aligned reads : 95
2025-11-11T04:58:30.963920Z  INFO oarfish::bulk: number of unique alignments : 82
2025-11-11T04:58:30.964537Z  INFO oarfish: oarfish completed successfully.
2025-11-11T04:58:30.971998Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T04:58:30.972429Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b63bb8d303/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T04:58:30.972440Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T04:58:30.972445Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T04:58:30.972506Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T04:58:30.972512Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T04:58:30.975321Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-11-11T04:58:30.975522Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-11-11T04:58:30.975570Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-11-11T04:58:30.975573Z  INFO oarfish::bulk: number of aligned reads : 179
2025-11-11T04:58:30.975575Z  INFO oarfish::bulk: number of unique alignments : 143
2025-11-11T04:58:30.976432Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b62090d4cd/config_file_1921462.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Nov 10 23:58:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample1_align2genome.bam
sample2 ->/tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample2_align2genome.bam
sample3 ->/tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Nov 10 23:58:52 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 @ Mon Nov 10 23:59:12 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 10 23:59:31 2025 ----------
2025-11-11T04:59:31.707253Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T04:59:31.707770Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T04:59:31.707795Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T04:59:31.707798Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T04:59:31.707854Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T04:59:31.707860Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T04:59:31.709709Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T04:59:31.709870Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-11-11T04:59:31.709900Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-11-11T04:59:31.709902Z  INFO oarfish::bulk: number of aligned reads : 96
2025-11-11T04:59:31.709905Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-11T04:59:31.710569Z  INFO oarfish: oarfish completed successfully.
2025-11-11T04:59:31.722396Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T04:59:31.722766Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T04:59:31.722775Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T04:59:31.722778Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T04:59:31.722834Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T04:59:31.722840Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T04:59:31.724463Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T04:59:31.724595Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-11-11T04:59:31.724620Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-11-11T04:59:31.724635Z  INFO oarfish::bulk: number of aligned reads : 95
2025-11-11T04:59:31.724637Z  INFO oarfish::bulk: number of unique alignments : 82
2025-11-11T04:59:31.725292Z  INFO oarfish: oarfish completed successfully.
2025-11-11T04:59:31.736734Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T04:59:31.737125Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b62090d4cd/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T04:59:31.737137Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T04:59:31.737141Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T04:59:31.737195Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T04:59:31.737201Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T04:59:31.739930Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-11-11T04:59:31.740090Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-11-11T04:59:31.740126Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-11-11T04:59:31.740128Z  INFO oarfish::bulk: number of aligned reads : 179
2025-11-11T04:59:31.740131Z  INFO oarfish::bulk: number of unique alignments : 143
2025-11-11T04:59:31.740840Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b6145b556f/config_file_1921462.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Nov 10 23:59:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Nov 10 23:59:32 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 @ Mon Nov 10 23:59:51 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Nov 10 23:59:51 2025 ----------
23:59:51 Mon Nov 10 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/Rtmpv8qU1Q/file1d51b629aa5e51/config_file_1921462.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Mon Nov 10 23:59:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample1_align2genome.bam
sample2 ->/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample2_align2genome.bam
sample3 ->/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Nov 11 00:00:13 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 @ Tue Nov 11 00:00:31 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:00:50 2025 ----------
00:00:50 Tue Nov 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample1_realign2transcript.bam', '/tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample2_realign2transcript.bam', '/tmp/Rtmpv8qU1Q/file1d51b6145b556f/sample3_realign2transcript.bam'] /tmp/Rtmpv8qU1Q/file1d51b6145b556f/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/Rtmpv8qU1Q/file1d51b661f52274/config_file_1921462.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 @ Tue Nov 11 00:00:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Nov 11 00:00:52 2025 -------------
Inputs:  ['/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample1_realign2transcript.bam', '/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample2_realign2transcript.bam', '/tmp/Rtmpv8qU1Q/file1d51b629aa5e51/sample3_realign2transcript.bam'] /tmp/Rtmpv8qU1Q/file1d51b629aa5e51/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 @ Tue Nov 11 00:00:52 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Tue Nov 11 00:00:53 2025 ----------
2025-11-11T05:00:53.364696Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:00:53.365042Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-11-11T05:00:53.365052Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:00:53.365055Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:00:53.365131Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:00:53.365139Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-11T05:00:53.367702Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T05:00:53.367824Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-11-11T05:00:53.367843Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-11-11T05:00:53.367846Z  INFO oarfish::bulk: number of aligned reads : 98
2025-11-11T05:00:53.367848Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-11T05:00:53.368494Z  INFO oarfish: oarfish completed successfully.
2025-11-11T05:00:53.375564Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:00:53.375906Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-11-11T05:00:53.375914Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:00:53.375917Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:00:53.375980Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:00:53.375986Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-11T05:00:53.378582Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T05:00:53.378713Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-11-11T05:00:53.378737Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-11-11T05:00:53.378740Z  INFO oarfish::bulk: number of aligned reads : 97
2025-11-11T05:00:53.378742Z  INFO oarfish::bulk: number of unique alignments : 79
2025-11-11T05:00:53.379379Z  INFO oarfish: oarfish completed successfully.
2025-11-11T05:00:53.386403Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:00:53.386749Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b661f52274/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-11-11T05:00:53.386757Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:00:53.386759Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:00:53.386824Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:00:53.386830Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-11T05:00:53.391089Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T05:00:53.391310Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-11-11T05:00:53.391340Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-11-11T05:00:53.391343Z  INFO oarfish::bulk: number of aligned reads : 187
2025-11-11T05:00:53.391345Z  INFO oarfish::bulk: number of unique alignments : 140
2025-11-11T05:00:53.392039Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b62cc93747/config_file_1921462.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 @ Tue Nov 11 00:00:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample1_align2genome.bam
sample2 ->/tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample2_align2genome.bam
sample3 ->/tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Nov 11 00:01:12 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:01:12 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:01:30 2025 ----------
2025-11-11T05:01:30.864875Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:01:30.865240Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-11-11T05:01:30.865253Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:01:30.865272Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:01:30.865338Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:01:30.865345Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-11T05:01:30.867936Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T05:01:30.868065Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-11-11T05:01:30.868091Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-11-11T05:01:30.868093Z  INFO oarfish::bulk: number of aligned reads : 98
2025-11-11T05:01:30.868096Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-11T05:01:30.868735Z  INFO oarfish: oarfish completed successfully.
2025-11-11T05:01:30.877686Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:01:30.878040Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-11-11T05:01:30.878048Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:01:30.878051Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:01:30.878125Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:01:30.878135Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-11T05:01:30.880646Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T05:01:30.880770Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-11-11T05:01:30.880793Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-11-11T05:01:30.880795Z  INFO oarfish::bulk: number of aligned reads : 97
2025-11-11T05:01:30.880809Z  INFO oarfish::bulk: number of unique alignments : 79
2025-11-11T05:01:30.881418Z  INFO oarfish: oarfish completed successfully.
2025-11-11T05:01:30.890265Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:01:30.890636Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b62cc93747/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-11-11T05:01:30.890643Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:01:30.890646Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:01:30.890716Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:01:30.890722Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-11T05:01:30.895038Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-11T05:01:30.895260Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-11-11T05:01:30.895291Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-11-11T05:01:30.895294Z  INFO oarfish::bulk: number of aligned reads : 187
2025-11-11T05:01:30.895296Z  INFO oarfish::bulk: number of unique alignments : 140
2025-11-11T05:01:30.895967Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/config_file_1921462.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 @ Tue Nov 11 00:01:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Nov 11 00:01:32 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:01:32 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Nov 11 00:01:33 2025 ----------
00:01:33 Tue Nov 11 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/Rtmpv8qU1Q/file1d51b66ad40c0a/config_file_1921462.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 @ Tue Nov 11 00:01:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample1_align2genome.bam
sample2 ->/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample2_align2genome.bam
sample3 ->/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Nov 11 00:01:54 2025 -------------
Inputs:  ['/tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample1_realign2transcript.bam', '/tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample2_realign2transcript.bam', '/tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/sample3_realign2transcript.bam'] /tmp/Rtmpv8qU1Q/file1d51b656fcc5f1/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 @ Tue Nov 11 00:01:54 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:02:12 2025 ----------
00:02:12 Tue Nov 11 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/Rtmpv8qU1Q/file1d51b65a27a7a/config_file_1921462.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 @ Tue Nov 11 00:02:13 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b65a27a7a/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 @ Tue Nov 11 00:02:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b65a27a7a/matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b65a27a7a/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Nov 11 00:02:14 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 @ Tue Nov 11 00:02:23 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/Rtmpv8qU1Q/file1d51b65a27a7a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b65a27a7a/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpv8qU1Q/file1d51b65a27a7a/matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b65a27a7a/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 @ Tue Nov 11 00:02:23 2025 ----------
2025-11-11T05:02:23.974085Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:02:23.974490Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b65a27a7a/realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:02:23.974502Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:02:23.974506Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:02:23.974564Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:02:23.974570Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T05:02:23.980587Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b66a6f8259/config_file_1921462.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 @ Tue Nov 11 00:02:24 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 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b66a6f8259/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 @ Tue Nov 11 00:02:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b66a6f8259/matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b66a6f8259/align2genome.bam
-- Running step: isoform_identification @ Tue Nov 11 00:02:42 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 @ Tue Nov 11 00:02:52 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b66a6f8259/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b66a6f8259/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b66a6f8259/matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b66a6f8259/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:03:10 2025 ----------
2025-11-11T05:03:10.988143Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:03:10.988587Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b66a6f8259/realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:03:10.988599Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:03:10.988604Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:03:10.988666Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:03:10.988674Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T05:03:10.995328Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b62d511e55/config_file_1921462.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 @ Tue Nov 11 00:03:11 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b62d511e55/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 @ Tue Nov 11 00:03:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b62d511e55/matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b62d511e55/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 @ Tue Nov 11 00:03:12 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 @ Tue Nov 11 00:03:22 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/Rtmpv8qU1Q/file1d51b62d511e55/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b62d511e55/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpv8qU1Q/file1d51b62d511e55/matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b62d511e55/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 @ Tue Nov 11 00:03:22 2025 ----------
00:03:22 Tue Nov 11 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample1_realign2transcript.bam', '/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample2_realign2transcript.bam', '/tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/sample3_realign2transcript.bam'] /tmp/Rtmpv8qU1Q/file1d51b66ad40c0a/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/Rtmpv8qU1Q/file1d51b6524a7011/config_file_1921462.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 @ Tue Nov 11 00:03:23 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/Rtmpv8qU1Q/file1d51b6524a7011/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 @ Tue Nov 11 00:03:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b6524a7011/matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6524a7011/align2genome.bam
-- Running step: isoform_identification @ Tue Nov 11 00:03:42 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 @ Tue Nov 11 00:03:52 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6524a7011/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6524a7011/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b6524a7011/matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6524a7011/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:04:10 2025 ----------
00:04:10 Tue Nov 11 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/Rtmpv8qU1Q/file1d51b64c545e65/config_file_1921462.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 @ Tue Nov 11 00:04:11 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b64c545e65/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 @ Tue Nov 11 00:04:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b64c545e65/matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b64c545e65/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Nov 11 00:04:11 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 @ Tue Nov 11 00:04:12 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b64c545e65/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b64c545e65/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpv8qU1Q/file1d51b64c545e65/matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b64c545e65/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 @ Tue Nov 11 00:04:12 2025 ----------
2025-11-11T05:04:12.322577Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:04:12.322983Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b64c545e65/realign2transcript.bam, contains 10 reference sequences.
2025-11-11T05:04:12.322991Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:04:12.322994Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:04:12.323066Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:04:12.323073Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-11T05:04:12.333662Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b6668dcda8/config_file_1921462.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 @ Tue Nov 11 00:04:13 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b6668dcda8/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 @ Tue Nov 11 00:04:13 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b6668dcda8/matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6668dcda8/align2genome.bam
-- Running step: isoform_identification @ Tue Nov 11 00:04:31 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:04:31 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/Rtmpv8qU1Q/file1d51b6668dcda8/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6668dcda8/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b6668dcda8/matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6668dcda8/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:04:49 2025 ----------
2025-11-11T05:04:49.555073Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:04:49.555515Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b6668dcda8/realign2transcript.bam, contains 10 reference sequences.
2025-11-11T05:04:49.555528Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:04:49.555531Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:04:49.555616Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:04:49.555624Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-11T05:04:49.566393Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b66f5ad30e/config_file_1921462.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 @ Tue Nov 11 00:04:50 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b66f5ad30e/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 @ Tue Nov 11 00:04:50 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b66f5ad30e/matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66f5ad30e/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 @ Tue Nov 11 00:04:50 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:04:51 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/Rtmpv8qU1Q/file1d51b66f5ad30e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b66f5ad30e/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpv8qU1Q/file1d51b66f5ad30e/matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66f5ad30e/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 @ Tue Nov 11 00:04:51 2025 ----------
00:04:51 Tue Nov 11 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b640bc9e06/config_file_1921462.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 @ Tue Nov 11 00:04:52 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/Rtmpv8qU1Q/file1d51b640bc9e06/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 @ Tue Nov 11 00:04:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b640bc9e06/matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b640bc9e06/align2genome.bam
-- Running step: isoform_identification @ Tue Nov 11 00:05:10 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:05:11 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/Rtmpv8qU1Q/file1d51b640bc9e06/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b640bc9e06/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b640bc9e06/matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b640bc9e06/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:05:29 2025 ----------
00:05:29 Tue Nov 11 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b625aa9666/config_file_1921462.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 @ Tue Nov 11 00:05:30 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/Rtmpv8qU1Q/file1d51b625aa9666/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b625aa9666/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b625aa9666/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b625aa9666/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/Rtmpv8qU1Q/file1d51b625aa9666/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b625aa9666/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/Rtmpv8qU1Q/file1d51b625aa9666/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b625aa9666/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/Rtmpv8qU1Q/file1d51b625aa9666/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b625aa9666/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 @ Tue Nov 11 00:05:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Nov 11 00:05:33 2025 ----------------
00:05:33 Tue Nov 11 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_align2genome.bam', and
'/tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.72gene_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, 411480.59Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.28gene_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, 1431503.07Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.28gene_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, 1361963.89Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.07gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 790602.43Read/s]
-- Running step: isoform_identification @ Tue Nov 11 00:05:34 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Tue Nov 11 00:05:58 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b625aa9666/fastq, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/fastq/sample1.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/fastq/sample2.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Tue Nov 11 00:05:59 2025 ----------
2025-11-11T05:05:59.235380Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:05:59.235832Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:05:59.235854Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:05:59.235857Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:05:59.235913Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:05:59.235919Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T05:05:59.242041Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-11T05:05:59.532655Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:05:59.533035Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:05:59.533042Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:05:59.533045Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:05:59.533095Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:05:59.533100Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T05:05:59.845208Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:05:59.845737Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:05:59.845745Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:05:59.845748Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:05:59.845806Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:05:59.845811Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T05:06:00.143773Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:06:00.144159Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b625aa9666/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:06:00.144170Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:06:00.144174Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:06:00.144228Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:06:00.144233Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/config_file_1921462.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 @ Tue Nov 11 00:06:00 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/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/Rtmpv8qU1Q/file1d51b6edf95f3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/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/Rtmpv8qU1Q/file1d51b6edf95f3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/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/Rtmpv8qU1Q/file1d51b6edf95f3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/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 @ Tue Nov 11 00:06:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Nov 11 00:06:20 2025 ----------------
00:06:20 Tue Nov 11 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_align2genome.bam', and
'/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_align2genome.bam'
parsing /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.37gene_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, 397700.07Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.02gene_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, 1475205.40Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.70gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1322790.46Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.16gene_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, 728380.11Read/s]
-- Running step: isoform_identification @ Tue Nov 11 00:06:21 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Tue Nov 11 00:06:45 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/fastq, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/fastq/sample1.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/fastq/sample2.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:07:04 2025 ----------
2025-11-11T05:07:04.090265Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:07:04.090671Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:07:04.090681Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:07:04.090685Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:07:04.090738Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:07:04.090745Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T05:07:04.096562Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-11T05:07:04.478916Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:07:04.479553Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:07:04.479565Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:07:04.479569Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:07:04.479641Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:07:04.479647Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T05:07:04.811939Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:07:04.812329Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:07:04.812340Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:07:04.812344Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:07:04.812405Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:07:04.812411Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-11T05:07:05.142067Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:07:05.142446Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b6edf95f3/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-11T05:07:05.142458Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:07:05.142462Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:07:05.142512Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:07:05.142530Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b67488e71d/config_file_1921462.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 @ Tue Nov 11 00:07:05 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b67488e71d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67488e71d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67488e71d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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 @ Tue Nov 11 00:07:06 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/sample1_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/sample2_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/sample3_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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 @ Tue Nov 11 00:07:08 2025 ----------------
00:07:08 Tue Nov 11 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample1_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample2_align2genome.bam', and
'/tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample3_align2genome.bam'
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 418760.38Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 41.16gene_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, 1355625.08Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.34gene_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, 1263040.23Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 667670.17Read/s]
-- Running step: isoform_identification @ Tue Nov 11 00:07:09 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
  |                                                                            
  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
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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 @ Tue Nov 11 00:07:31 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b67488e71d/fastq, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/fastq/sample1.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/fastq/sample2.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample1_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample2_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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/Rtmpv8qU1Q/file1d51b67488e71d/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b67488e71d/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 @ Tue Nov 11 00:07:32 2025 ----------
00:07:32 Tue Nov 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b67488e71d/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample1_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample2_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample3_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b67488e71d/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/config_file_1921462.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 @ Tue Nov 11 00:07:34 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/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/Rtmpv8qU1Q/file1d51b67a6215f1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/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/Rtmpv8qU1Q/file1d51b67a6215f1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/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/Rtmpv8qU1Q/file1d51b67a6215f1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/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 @ Tue Nov 11 00:07:35 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Nov 11 00:07:54 2025 ----------------
00:07:54 Tue Nov 11 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_align2genome.bam', and
'/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/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/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 387257.08Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.84gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1385356.06Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.82gene_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, 1339348.58Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.21gene_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, 707971.10Read/s]
-- Running step: isoform_identification @ Tue Nov 11 00:07:55 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Tue Nov 11 00:08:17 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/fastq, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/fastq/sample1.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/fastq/sample2.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:08:37 2025 ----------
00:08:37 Tue Nov 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b67a6215f1/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/config_file_1921462.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 @ Tue Nov 11 00:08:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/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/Rtmpv8qU1Q/file1d51b66cc4f04e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/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/Rtmpv8qU1Q/file1d51b66cc4f04e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/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/Rtmpv8qU1Q/file1d51b66cc4f04e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/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 @ Tue Nov 11 00:08:39 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample3_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Nov 11 00:08:41 2025 ----------------
00:08:41 Tue Nov 11 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_align2genome.bam', and
'/tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/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/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 393019.49Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 38.92gene_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, 1314169.70Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.22gene_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, 1250239.66Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.26gene_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, 626239.85Read/s]
-- Running step: isoform_identification @ Tue Nov 11 00:08:42 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:08:42 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/fastq, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/fastq/sample1.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/fastq/sample2.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Tue Nov 11 00:08:44 2025 ----------
2025-11-11T05:08:44.512624Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:08:44.513232Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-11-11T05:08:44.513242Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:08:44.513245Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:08:44.513327Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:08:44.513335Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-11T05:08:44.525153Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-11T05:08:45.099624Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:08:45.100007Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-11-11T05:08:45.100015Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:08:45.100019Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:08:45.100097Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:08:45.100104Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-11T05:08:45.669100Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:08:45.669612Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-11-11T05:08:45.669620Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:08:45.669623Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:08:45.669710Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:08:45.669717Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-11T05:08:46.240177Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:08:46.240662Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b66cc4f04e/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-11-11T05:08:46.240671Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:08:46.240674Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:08:46.240761Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:08:46.240769Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/config_file_1921462.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 @ Tue Nov 11 00:08:47 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/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/Rtmpv8qU1Q/file1d51b614a30d7b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/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/Rtmpv8qU1Q/file1d51b614a30d7b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/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/Rtmpv8qU1Q/file1d51b614a30d7b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/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 @ Tue Nov 11 00:08:47 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Nov 11 00:09:06 2025 ----------------
00:09:06 Tue Nov 11 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_align2genome.bam', and
'/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_align2genome.bam'
parsing /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.89gene_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, 378779.76Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.79gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1391422.51Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.85gene_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, 1223542.59Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.89gene_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, 677768.73Read/s]
-- Running step: isoform_identification @ Tue Nov 11 00:09:07 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:09:07 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/fastq, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/fastq/sample1.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/fastq/sample2.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:09:28 2025 ----------
2025-11-11T05:09:28.960316Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:09:28.960762Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-11-11T05:09:28.960788Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:09:28.960791Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:09:28.960882Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:09:28.960890Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-11T05:09:28.973558Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-11T05:09:29.627487Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:09:29.627920Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-11-11T05:09:29.627929Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:09:29.627932Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:09:29.628012Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:09:29.628019Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-11T05:09:30.288454Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:09:30.288915Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-11-11T05:09:30.288922Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:09:30.288925Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:09:30.288999Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:09:30.289006Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-11T05:09:30.863813Z  INFO oarfish: setting user-provided filter parameters.
2025-11-11T05:09:30.864179Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpv8qU1Q/file1d51b614a30d7b/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-11-11T05:09:30.864191Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-11T05:09:30.864193Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-11T05:09:30.864269Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-11T05:09:30.864277Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/config_file_1921462.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 @ Tue Nov 11 00:09:31 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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 @ Tue Nov 11 00:09:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_matched_reads.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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 @ Tue Nov 11 00:09:33 2025 ----------------
00:09:33 Tue Nov 11 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_align2genome.bam', and
'/tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_align2genome.bam'
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.58gene_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, 424026.85Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.57gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.56gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1198783.58Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1292145.41Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.74gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 710224.87Read/s]
-- Running step: isoform_identification @ Tue Nov 11 00:09:34 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:09:35 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/fastq, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/fastq/sample1.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/fastq/sample2.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/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 @ Tue Nov 11 00:09:36 2025 ----------
00:09:36 Tue Nov 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b6652e6f59/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpv8qU1Q/file1d51b6e17938f/config_file_1921462.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 @ Tue Nov 11 00:09:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpv8qU1Q/file1d51b6e17938f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6e17938f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6e17938f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6e17938f/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/Rtmpv8qU1Q/file1d51b6e17938f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6e17938f/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/Rtmpv8qU1Q/file1d51b6e17938f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6e17938f/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/Rtmpv8qU1Q/file1d51b6e17938f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpv8qU1Q/file1d51b6e17938f/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 @ Tue Nov 11 00:09:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_align2genome.bam
/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_matched_reads.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Nov 11 00:09:58 2025 ----------------
00:09:58 Tue Nov 11 2025 quantify genes 
Using BAM(s): '/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_align2genome.bam',
'/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_align2genome.bam', and
'/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.35gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 363155.78Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1386821.85Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.14gene_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, 1322290.04Read/s]
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.10gene_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, 747967.76Read/s]
-- Running step: isoform_identification @ Tue Nov 11 00:09:59 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Nov 11 00:10:00 2025 -------------------
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6e17938f/fastq, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/fastq/sample1.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/fastq/sample2.fq.gz, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_matched_reads.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_realign2transcript.bam
/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Nov 11 00:10:19 2025 ----------
00:10:19 Tue Nov 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_realign2transcript.bam...
parsing /tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpv8qU1Q/file1d51b6e17938f/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
> 
> proc.time()
   user  system elapsed 
727.282  43.410 757.801