##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
###
##############################################################################
##############################################################################


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

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     25.050  1.645  26.695
blaze                         4.298 17.741  12.455
find_variants                19.148  0.075  18.612
bulk_long_pipeline            2.290 13.266   2.406
sc_long_multisample_pipeline  7.848  6.312   7.981
sc_plot_genotype             10.934  0.357  10.141
MultiSampleSCPipeline        10.025  0.686  10.985
sc_DTU_analysis               6.966  2.455   7.121
plot_isoform_heatmap          7.488  0.754   8.241
create_sce_from_dir           3.426  2.451   3.598
sc_long_pipeline              3.136  1.920   2.786
* 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.

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


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

R version 4.5.1 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.

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623914dd4a81/config_file_2515513.json 
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623914dd4a81/config_file_2515513.json 
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623914dd4a81/config_file_2515513.json 
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662397309772b/config_file_2515513.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/RtmpW5VxCf/file2662395a4f8ba5/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/RtmpW5VxCf/file2662392f7e622/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/RtmpW5VxCf/file2662392f7e622/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623947b67042/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623947b67042/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623947b67042/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623947b67042/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/RtmpW5VxCf/file2662397a3e79ba/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/RtmpW5VxCf/file2662398c1290e/config_file_2515513.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Sat Oct 11 00:15:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpW5VxCf/file2662398c1290e/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpW5VxCf/file2662398c1290e/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpW5VxCf/file2662398c1290e/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 11 00:15:32 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[00:15:38] 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.

[00:15:38] 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.

[00:15:39] 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.

[00:15:39] 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.

[00:15:40] 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.

[00:15:41] 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 @ Sat Oct 11 00:15:59 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpW5VxCf/file2662398c1290e/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpW5VxCf/file2662398c1290e/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpW5VxCf/file2662398c1290e/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sat Oct 11 00:16:00 2025 ----------
2025-10-11T04:16:00.420921Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:16:00.421360Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662398c1290e/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:16:00.421372Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:16:00.421376Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:16:00.421435Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:16:00.421441Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:16:00.422997Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:16:00.423136Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T04:16:00.423158Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-11T04:16:00.423169Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-11T04:16:00.423171Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-11T04:16:00.423759Z  INFO oarfish: oarfish completed successfully.
2025-10-11T04:16:00.430702Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:16:00.431035Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662398c1290e/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:16:00.431043Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:16:00.431047Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:16:00.431106Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:16:00.431111Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:16:00.432667Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:16:00.432788Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T04:16:00.432812Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-11T04:16:00.432814Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-11T04:16:00.432816Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-11T04:16:00.433400Z  INFO oarfish: oarfish completed successfully.
2025-10-11T04:16:00.440276Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:16:00.440622Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662398c1290e/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:16:00.440630Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:16:00.440633Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:16:00.440683Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:16:00.440688Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:16:00.443371Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-11T04:16:00.443524Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-11T04:16:00.443564Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-11T04:16:00.443566Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-11T04:16:00.443568Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-11T04:16:00.444254Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623999dc76d/config_file_2515513.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Sat Oct 11 00:16:00 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpW5VxCf/file26623999dc76d/sample1_align2genome.bam
sample2 ->/tmp/RtmpW5VxCf/file26623999dc76d/sample2_align2genome.bam
sample3 ->/tmp/RtmpW5VxCf/file26623999dc76d/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct 11 00:16:20 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 @ Sat Oct 11 00:16:42 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpW5VxCf/file26623999dc76d/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpW5VxCf/file26623999dc76d/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpW5VxCf/file26623999dc76d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:17:02 2025 ----------
2025-10-11T04:17:02.254564Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:17:02.255079Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623999dc76d/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:17:02.255101Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:17:02.255104Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:17:02.255199Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:17:02.255207Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:17:02.257075Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:17:02.257226Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T04:17:02.257257Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-11T04:17:02.257259Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-11T04:17:02.257261Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-11T04:17:02.257860Z  INFO oarfish: oarfish completed successfully.
2025-10-11T04:17:02.269729Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:17:02.270090Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623999dc76d/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:17:02.270098Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:17:02.270101Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:17:02.270172Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:17:02.270182Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:17:02.271787Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:17:02.271917Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T04:17:02.271943Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-11T04:17:02.271958Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-11T04:17:02.271960Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-11T04:17:02.272562Z  INFO oarfish: oarfish completed successfully.
2025-10-11T04:17:02.283812Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:17:02.284320Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623999dc76d/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:17:02.284331Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:17:02.284334Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:17:02.284389Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:17:02.284394Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:17:02.287553Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-11T04:17:02.287703Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-11T04:17:02.287731Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-11T04:17:02.287734Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-11T04:17:02.287736Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-11T04:17:02.288456Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662391263b115/config_file_2515513.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Sat Oct 11 00:17:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpW5VxCf/file2662391263b115/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpW5VxCf/file2662391263b115/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpW5VxCf/file2662391263b115/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 11 00:17:03 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 @ Sat Oct 11 00:17:22 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpW5VxCf/file2662391263b115/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpW5VxCf/file2662391263b115/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpW5VxCf/file2662391263b115/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 11 00:17:23 2025 ----------
00:17:23 Sat Oct 11 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/RtmpW5VxCf/file2662392da026f7/config_file_2515513.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Sat Oct 11 00:17:24 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpW5VxCf/file2662392da026f7/sample1_align2genome.bam
sample2 ->/tmp/RtmpW5VxCf/file2662392da026f7/sample2_align2genome.bam
sample3 ->/tmp/RtmpW5VxCf/file2662392da026f7/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct 11 00:17:42 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 @ Sat Oct 11 00:18:02 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpW5VxCf/file2662392da026f7/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpW5VxCf/file2662392da026f7/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpW5VxCf/file2662392da026f7/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:18:20 2025 ----------
00:18:20 Sat Oct 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpW5VxCf/file2662391263b115/sample1_realign2transcript.bam', '/tmp/RtmpW5VxCf/file2662391263b115/sample2_realign2transcript.bam', '/tmp/RtmpW5VxCf/file2662391263b115/sample3_realign2transcript.bam'] /tmp/RtmpW5VxCf/file2662391263b115/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/RtmpW5VxCf/file2662392b6014fb/config_file_2515513.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Sat Oct 11 00:18:21 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpW5VxCf/file2662392b6014fb/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpW5VxCf/file2662392b6014fb/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpW5VxCf/file2662392b6014fb/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 11 00:18:22 2025 -------------
Inputs:  ['/tmp/RtmpW5VxCf/file2662392da026f7/sample1_realign2transcript.bam', '/tmp/RtmpW5VxCf/file2662392da026f7/sample2_realign2transcript.bam', '/tmp/RtmpW5VxCf/file2662392da026f7/sample3_realign2transcript.bam'] /tmp/RtmpW5VxCf/file2662392da026f7/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 @ Sat Oct 11 00:18:22 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpW5VxCf/file2662392b6014fb/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpW5VxCf/file2662392b6014fb/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpW5VxCf/file2662392b6014fb/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sat Oct 11 00:18:23 2025 ----------
2025-10-11T04:18:23.497377Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:18:23.497851Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662392b6014fb/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T04:18:23.497862Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:18:23.497866Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:18:23.497938Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:18:23.497946Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T04:18:23.500616Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:18:23.500745Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T04:18:23.500766Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-11T04:18:23.500769Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-11T04:18:23.500771Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-11T04:18:23.501396Z  INFO oarfish: oarfish completed successfully.
2025-10-11T04:18:23.508823Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:18:23.509183Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662392b6014fb/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T04:18:23.509194Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:18:23.509198Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:18:23.509272Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:18:23.509278Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T04:18:23.512010Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:18:23.512172Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T04:18:23.512205Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-11T04:18:23.512208Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-11T04:18:23.512210Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-11T04:18:23.512830Z  INFO oarfish: oarfish completed successfully.
2025-10-11T04:18:23.520576Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:18:23.521012Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662392b6014fb/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T04:18:23.521020Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:18:23.521023Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:18:23.521095Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:18:23.521102Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T04:18:23.525461Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:18:23.525606Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-11T04:18:23.525633Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-11T04:18:23.525635Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-11T04:18:23.525638Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-11T04:18:23.526333Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623949bf3472/config_file_2515513.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Sat Oct 11 00:18:23 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpW5VxCf/file26623949bf3472/sample1_align2genome.bam
sample2 ->/tmp/RtmpW5VxCf/file26623949bf3472/sample2_align2genome.bam
sample3 ->/tmp/RtmpW5VxCf/file26623949bf3472/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct 11 00:18:41 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:18:42 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpW5VxCf/file26623949bf3472/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpW5VxCf/file26623949bf3472/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpW5VxCf/file26623949bf3472/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:19:00 2025 ----------
2025-10-11T04:19:00.081302Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:19:00.081666Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623949bf3472/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T04:19:00.081677Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:19:00.081692Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:19:00.081768Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:19:00.081775Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T04:19:00.084475Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:19:00.084607Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T04:19:00.084627Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-11T04:19:00.084629Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-11T04:19:00.084631Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-11T04:19:00.085263Z  INFO oarfish: oarfish completed successfully.
2025-10-11T04:19:00.093235Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:19:00.093693Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623949bf3472/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T04:19:00.093701Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:19:00.093704Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:19:00.093768Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:19:00.093774Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T04:19:00.096355Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:19:00.096467Z  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    │
╰─────────────────────────────────┴───────╯

2025-10-11T04:19:00.096490Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-11T04:19:00.096492Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-11T04:19:00.096502Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-11T04:19:00.097076Z  INFO oarfish: oarfish completed successfully.
2025-10-11T04:19:00.104790Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:19:00.105139Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623949bf3472/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-11T04:19:00.105150Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:19:00.105153Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:19:00.105220Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:19:00.105227Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T04:19:00.109536Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-11T04:19:00.109732Z  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   │
╰─────────────────────────────────┴───────╯

2025-10-11T04:19:00.109759Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-11T04:19:00.109761Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-11T04:19:00.109764Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-11T04:19:00.110464Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662399875abb/config_file_2515513.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Sat Oct 11 00:19:00 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpW5VxCf/file2662399875abb/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpW5VxCf/file2662399875abb/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpW5VxCf/file2662399875abb/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 11 00:19:01 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:19:01 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpW5VxCf/file2662399875abb/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpW5VxCf/file2662399875abb/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpW5VxCf/file2662399875abb/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 11 00:19:02 2025 ----------
00:19:02 Sat Oct 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/RtmpW5VxCf/file266239645f008a/config_file_2515513.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Sat Oct 11 00:19:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpW5VxCf/file266239645f008a/sample1_align2genome.bam
sample2 ->/tmp/RtmpW5VxCf/file266239645f008a/sample2_align2genome.bam
sample3 ->/tmp/RtmpW5VxCf/file266239645f008a/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Oct 11 00:19:21 2025 -------------
Inputs:  ['/tmp/RtmpW5VxCf/file2662399875abb/sample1_realign2transcript.bam', '/tmp/RtmpW5VxCf/file2662399875abb/sample2_realign2transcript.bam', '/tmp/RtmpW5VxCf/file2662399875abb/sample3_realign2transcript.bam'] /tmp/RtmpW5VxCf/file2662399875abb/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 @ Sat Oct 11 00:19:22 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpW5VxCf/file266239645f008a/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpW5VxCf/file266239645f008a/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpW5VxCf/file266239645f008a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:19:40 2025 ----------
00:19:40 Sat Oct 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/RtmpW5VxCf/file266239a59980e/config_file_2515513.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 @ Sat Oct 11 00:19:41 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/RtmpW5VxCf/file266239a59980e/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 @ Sat Oct 11 00:19:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpW5VxCf/file266239a59980e/matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file266239a59980e/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 11 00:19:41 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 @ Sat Oct 11 00:19:52 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/RtmpW5VxCf/file266239a59980e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file266239a59980e/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpW5VxCf/file266239a59980e/matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file266239a59980e/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Sat Oct 11 00:19:52 2025 ----------
2025-10-11T04:19:52.221009Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:19:52.221459Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file266239a59980e/realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:19:52.221472Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:19:52.221476Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:19:52.221537Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:19:52.221544Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:19:52.228015Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662394f23ea2d/config_file_2515513.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 @ Sat Oct 11 00:19:52 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/RtmpW5VxCf/file2662394f23ea2d/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 @ Sat Oct 11 00:19:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662394f23ea2d/matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662394f23ea2d/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 11 00:20:11 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 @ Sat Oct 11 00:20:21 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/RtmpW5VxCf/file2662394f23ea2d/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662394f23ea2d/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662394f23ea2d/matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662394f23ea2d/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:20:38 2025 ----------
2025-10-11T04:20:38.602036Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:20:38.602489Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662394f23ea2d/realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:20:38.602500Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:20:38.602504Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:20:38.602561Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:20:38.602568Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:20:38.609188Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623951aa52b6/config_file_2515513.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 @ Sat Oct 11 00:20:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpW5VxCf/file26623951aa52b6/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 @ Sat Oct 11 00:20:39 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpW5VxCf/file26623951aa52b6/matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623951aa52b6/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 @ Sat Oct 11 00:20:39 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 @ Sat Oct 11 00:20:50 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623951aa52b6/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623951aa52b6/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpW5VxCf/file26623951aa52b6/matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623951aa52b6/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 11 00:20:50 2025 ----------
00:20:50 Sat Oct 11 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpW5VxCf/file266239645f008a/sample1_realign2transcript.bam', '/tmp/RtmpW5VxCf/file266239645f008a/sample2_realign2transcript.bam', '/tmp/RtmpW5VxCf/file266239645f008a/sample3_realign2transcript.bam'] /tmp/RtmpW5VxCf/file266239645f008a/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/RtmpW5VxCf/file2662393c26e8cf/config_file_2515513.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 @ Sat Oct 11 00:20:51 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpW5VxCf/file2662393c26e8cf/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 @ Sat Oct 11 00:20:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662393c26e8cf/matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c26e8cf/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 11 00:21:10 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 @ Sat Oct 11 00:21:21 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/RtmpW5VxCf/file2662393c26e8cf/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662393c26e8cf/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662393c26e8cf/matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c26e8cf/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:21:39 2025 ----------
00:21:39 Sat Oct 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/RtmpW5VxCf/file26623965c15bff/config_file_2515513.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 @ Sat Oct 11 00:21:40 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/RtmpW5VxCf/file26623965c15bff/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 @ Sat Oct 11 00:21:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpW5VxCf/file26623965c15bff/matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623965c15bff/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Oct 11 00:21:40 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 @ Sat Oct 11 00:21:41 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/RtmpW5VxCf/file26623965c15bff/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623965c15bff/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpW5VxCf/file26623965c15bff/matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623965c15bff/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Sat Oct 11 00:21:41 2025 ----------
2025-10-11T04:21:41.337670Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:21:41.338140Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623965c15bff/realign2transcript.bam, contains 10 reference sequences.
2025-10-11T04:21:41.338152Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:21:41.338155Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:21:41.338226Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:21:41.338233Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T04:21:41.347829Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623949cc2f25/config_file_2515513.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 @ Sat Oct 11 00:21:42 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/RtmpW5VxCf/file26623949cc2f25/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 @ Sat Oct 11 00:21:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpW5VxCf/file26623949cc2f25/matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file26623949cc2f25/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 11 00:21:59 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:21:59 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623949cc2f25/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623949cc2f25/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpW5VxCf/file26623949cc2f25/matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file26623949cc2f25/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:22:17 2025 ----------
2025-10-11T04:22:17.290474Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:22:17.290962Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623949cc2f25/realign2transcript.bam, contains 10 reference sequences.
2025-10-11T04:22:17.290972Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:22:17.290976Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:22:17.291058Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:22:17.291065Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-11T04:22:17.300443Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662391f6199e2/config_file_2515513.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 @ Sat Oct 11 00:22:18 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpW5VxCf/file2662391f6199e2/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 @ Sat Oct 11 00:22:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpW5VxCf/file2662391f6199e2/matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662391f6199e2/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 @ Sat Oct 11 00:22:18 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:22:18 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/RtmpW5VxCf/file2662391f6199e2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662391f6199e2/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpW5VxCf/file2662391f6199e2/matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662391f6199e2/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 11 00:22:19 2025 ----------
00:22:19 Sat Oct 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/RtmpW5VxCf/file26623910f2b43/config_file_2515513.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 @ Sat Oct 11 00:22:20 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/RtmpW5VxCf/file26623910f2b43/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 @ Sat Oct 11 00:22:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpW5VxCf/file26623910f2b43/matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file26623910f2b43/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 11 00:22:37 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:22:38 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/RtmpW5VxCf/file26623910f2b43/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623910f2b43/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpW5VxCf/file26623910f2b43/matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file26623910f2b43/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:22:56 2025 ----------
00:22:56 Sat Oct 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/RtmpW5VxCf/file26623970a72e17/config_file_2515513.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 11 00:22:57 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpW5VxCf/file26623970a72e17/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623970a72e17/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623970a72e17/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623970a72e17/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/RtmpW5VxCf/file26623970a72e17/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623970a72e17/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/RtmpW5VxCf/file26623970a72e17/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623970a72e17/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/RtmpW5VxCf/file26623970a72e17/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623970a72e17/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 @ Sat Oct 11 00:22:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpW5VxCf/file26623970a72e17/sampleA_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623970a72e17/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpW5VxCf/file26623970a72e17/sample1_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623970a72e17/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpW5VxCf/file26623970a72e17/sample2_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623970a72e17/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpW5VxCf/file26623970a72e17/sample3_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623970a72e17/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 11 00:22:59 2025 ----------------
00:22:59 Sat Oct 11 2025 quantify genes 
Using BAM(s): '/tmp/RtmpW5VxCf/file26623970a72e17/sampleA_align2genome.bam',
'/tmp/RtmpW5VxCf/file26623970a72e17/sample1_align2genome.bam',
'/tmp/RtmpW5VxCf/file26623970a72e17/sample2_align2genome.bam', and
'/tmp/RtmpW5VxCf/file26623970a72e17/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpW5VxCf/file26623970a72e17/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.74gene_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, 389573.49Read/s]
parsing /tmp/RtmpW5VxCf/file26623970a72e17/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.70gene_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, 1399967.96Read/s]
parsing /tmp/RtmpW5VxCf/file26623970a72e17/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.92gene_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, 1291986.20Read/s]
parsing /tmp/RtmpW5VxCf/file26623970a72e17/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.59gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 791079.59Read/s]
-- Running step: isoform_identification @ Sat Oct 11 00:23:01 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 @ Sat Oct 11 00:23:25 2025 -------------------
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623970a72e17/fastq, /tmp/RtmpW5VxCf/file26623970a72e17/fastq/sample1.fq.gz, /tmp/RtmpW5VxCf/file26623970a72e17/fastq/sample2.fq.gz, /tmp/RtmpW5VxCf/file26623970a72e17/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623970a72e17/sampleA_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623970a72e17/sample1_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623970a72e17/sample2_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623970a72e17/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623970a72e17/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623970a72e17/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623970a72e17/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623970a72e17/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpW5VxCf/file26623970a72e17/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file26623970a72e17/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpW5VxCf/file26623970a72e17/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file26623970a72e17/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpW5VxCf/file26623970a72e17/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file26623970a72e17/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpW5VxCf/file26623970a72e17/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file26623970a72e17/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Oct 11 00:23:26 2025 ----------
2025-10-11T04:23:26.687654Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:23:26.688188Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623970a72e17/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:23:26.688210Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:23:26.688213Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:23:26.688266Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:23:26.688271Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:23:26.693951Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-11T04:23:26.965667Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:23:26.966130Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623970a72e17/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:23:26.966142Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:23:26.966145Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:23:26.966204Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:23:26.966210Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:23:27.242334Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:23:27.242678Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623970a72e17/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:23:27.242686Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:23:27.242689Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:23:27.242750Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:23:27.242756Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:23:27.526854Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:23:27.527310Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623970a72e17/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:23:27.527323Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:23:27.527326Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:23:27.527376Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:23:27.527381Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623916a318f8/config_file_2515513.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 11 00:23:28 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpW5VxCf/file26623916a318f8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623916a318f8/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623916a318f8/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623916a318f8/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/RtmpW5VxCf/file26623916a318f8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623916a318f8/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/RtmpW5VxCf/file26623916a318f8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623916a318f8/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/RtmpW5VxCf/file26623916a318f8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623916a318f8/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 @ Sat Oct 11 00:23:28 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpW5VxCf/file26623916a318f8/sampleA_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file26623916a318f8/sampleA_align2genome.bam
/tmp/RtmpW5VxCf/file26623916a318f8/sample1_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file26623916a318f8/sample1_align2genome.bam
/tmp/RtmpW5VxCf/file26623916a318f8/sample2_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file26623916a318f8/sample2_align2genome.bam
/tmp/RtmpW5VxCf/file26623916a318f8/sample3_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file26623916a318f8/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 11 00:23:47 2025 ----------------
00:23:47 Sat Oct 11 2025 quantify genes 
Using BAM(s): '/tmp/RtmpW5VxCf/file26623916a318f8/sampleA_align2genome.bam',
'/tmp/RtmpW5VxCf/file26623916a318f8/sample1_align2genome.bam',
'/tmp/RtmpW5VxCf/file26623916a318f8/sample2_align2genome.bam', and
'/tmp/RtmpW5VxCf/file26623916a318f8/sample3_align2genome.bam'
parsing /tmp/RtmpW5VxCf/file26623916a318f8/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.26gene_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, 410273.10Read/s]
parsing /tmp/RtmpW5VxCf/file26623916a318f8/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.76gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1416611.73Read/s]
parsing /tmp/RtmpW5VxCf/file26623916a318f8/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.72gene_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, 1243862.40Read/s]
parsing /tmp/RtmpW5VxCf/file26623916a318f8/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.58gene_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, 743143.87Read/s]
-- Running step: isoform_identification @ Sat Oct 11 00:23:48 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 @ Sat Oct 11 00:24:13 2025 -------------------
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623916a318f8/fastq, /tmp/RtmpW5VxCf/file26623916a318f8/fastq/sample1.fq.gz, /tmp/RtmpW5VxCf/file26623916a318f8/fastq/sample2.fq.gz, /tmp/RtmpW5VxCf/file26623916a318f8/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623916a318f8/sampleA_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623916a318f8/sample1_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623916a318f8/sample2_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623916a318f8/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623916a318f8/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623916a318f8/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623916a318f8/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623916a318f8/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpW5VxCf/file26623916a318f8/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file26623916a318f8/sampleA_realign2transcript.bam
/tmp/RtmpW5VxCf/file26623916a318f8/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file26623916a318f8/sample1_realign2transcript.bam
/tmp/RtmpW5VxCf/file26623916a318f8/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file26623916a318f8/sample2_realign2transcript.bam
/tmp/RtmpW5VxCf/file26623916a318f8/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file26623916a318f8/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:24:30 2025 ----------
2025-10-11T04:24:30.892025Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:24:30.892447Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623916a318f8/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:24:30.892458Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:24:30.892462Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:24:30.892519Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:24:30.892525Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:24:30.898423Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-11T04:24:31.220663Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:24:31.221101Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623916a318f8/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:24:31.221112Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:24:31.221124Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:24:31.221181Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:24:31.221188Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:24:31.528426Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:24:31.528930Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623916a318f8/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:24:31.528940Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:24:31.528943Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:24:31.529001Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:24:31.529007Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-11T04:24:31.845195Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:24:31.845624Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file26623916a318f8/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-11T04:24:31.845635Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:24:31.845639Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:24:31.845702Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:24:31.845719Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662394e9e6397/config_file_2515513.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 11 00:24:32 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpW5VxCf/file2662394e9e6397/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662394e9e6397/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662394e9e6397/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662394e9e6397/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 @ Sat Oct 11 00:24:33 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/sample1_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/sample2_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/sample3_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662394e9e6397/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 11 00:24:34 2025 ----------------
00:24:34 Sat Oct 11 2025 quantify genes 
Using BAM(s): '/tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662394e9e6397/sample1_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662394e9e6397/sample2_align2genome.bam', and
'/tmp/RtmpW5VxCf/file2662394e9e6397/sample3_align2genome.bam'
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.02gene_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, 389240.88Read/s]
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.69gene_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, 1448709.59Read/s]
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.70gene_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, 1397728.61Read/s]
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 779842.33Read/s]
-- Running step: isoform_identification @ Sat Oct 11 00:24:35 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 @ Sat Oct 11 00:25:00 2025 -------------------
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662394e9e6397/fastq, /tmp/RtmpW5VxCf/file2662394e9e6397/fastq/sample1.fq.gz, /tmp/RtmpW5VxCf/file2662394e9e6397/fastq/sample2.fq.gz, /tmp/RtmpW5VxCf/file2662394e9e6397/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662394e9e6397/sample1_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662394e9e6397/sample2_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662394e9e6397/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662394e9e6397/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662394e9e6397/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662394e9e6397/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file2662394e9e6397/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/RtmpW5VxCf/file2662394e9e6397/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file2662394e9e6397/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 11 00:25:01 2025 ----------
00:25:01 Sat Oct 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662394e9e6397/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample1_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662394e9e6397/sample1_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample2_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662394e9e6397/sample2_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample3_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662394e9e6397/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662394e9e6397/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662393c548768/config_file_2515513.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 11 00:25:03 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/RtmpW5VxCf/file2662393c548768/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662393c548768/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662393c548768/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662393c548768/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/RtmpW5VxCf/file2662393c548768/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662393c548768/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/RtmpW5VxCf/file2662393c548768/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662393c548768/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/RtmpW5VxCf/file2662393c548768/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662393c548768/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 @ Sat Oct 11 00:25:03 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662393c548768/sampleA_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c548768/sampleA_align2genome.bam
/tmp/RtmpW5VxCf/file2662393c548768/sample1_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c548768/sample1_align2genome.bam
/tmp/RtmpW5VxCf/file2662393c548768/sample2_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c548768/sample2_align2genome.bam
/tmp/RtmpW5VxCf/file2662393c548768/sample3_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c548768/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 11 00:25:22 2025 ----------------
00:25:22 Sat Oct 11 2025 quantify genes 
Using BAM(s): '/tmp/RtmpW5VxCf/file2662393c548768/sampleA_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662393c548768/sample1_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662393c548768/sample2_align2genome.bam', and
'/tmp/RtmpW5VxCf/file2662393c548768/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/RtmpW5VxCf/file2662393c548768/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 19.03gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 360100.28Read/s]
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.53gene_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, 1428188.50Read/s]
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1347957.32Read/s]
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample3_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 702281.16Read/s]
-- Running step: isoform_identification @ Sat Oct 11 00:25:23 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 @ Sat Oct 11 00:25:48 2025 -------------------
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662393c548768/fastq, /tmp/RtmpW5VxCf/file2662393c548768/fastq/sample1.fq.gz, /tmp/RtmpW5VxCf/file2662393c548768/fastq/sample2.fq.gz, /tmp/RtmpW5VxCf/file2662393c548768/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662393c548768/sampleA_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662393c548768/sample1_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662393c548768/sample2_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662393c548768/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662393c548768/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662393c548768/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662393c548768/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662393c548768/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662393c548768/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c548768/sampleA_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662393c548768/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c548768/sample1_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662393c548768/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c548768/sample2_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662393c548768/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662393c548768/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:26:06 2025 ----------
00:26:06 Sat Oct 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpW5VxCf/file2662393c548768/sampleA_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662393c548768/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662393c548768/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample1_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662393c548768/sample1_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample2_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662393c548768/sample2_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample3_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662393c548768/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662393c548768/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662394097f1dc/config_file_2515513.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 11 00:26:08 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/RtmpW5VxCf/file2662394097f1dc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662394097f1dc/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662394097f1dc/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662394097f1dc/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/RtmpW5VxCf/file2662394097f1dc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662394097f1dc/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/RtmpW5VxCf/file2662394097f1dc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662394097f1dc/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/RtmpW5VxCf/file2662394097f1dc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662394097f1dc/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 @ Sat Oct 11 00:26:09 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpW5VxCf/file2662394097f1dc/sampleA_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662394097f1dc/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpW5VxCf/file2662394097f1dc/sample1_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662394097f1dc/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpW5VxCf/file2662394097f1dc/sample2_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662394097f1dc/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpW5VxCf/file2662394097f1dc/sample3_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file2662394097f1dc/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 11 00:26:10 2025 ----------------
00:26:10 Sat Oct 11 2025 quantify genes 
Using BAM(s): '/tmp/RtmpW5VxCf/file2662394097f1dc/sampleA_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662394097f1dc/sample1_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662394097f1dc/sample2_align2genome.bam', and
'/tmp/RtmpW5VxCf/file2662394097f1dc/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/RtmpW5VxCf/file2662394097f1dc/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 407182.35Read/s]
parsing /tmp/RtmpW5VxCf/file2662394097f1dc/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.37gene_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, 1396053.79Read/s]
parsing /tmp/RtmpW5VxCf/file2662394097f1dc/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.86gene_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, 1165213.91Read/s]
parsing /tmp/RtmpW5VxCf/file2662394097f1dc/sample3_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 717367.45Read/s]
-- Running step: isoform_identification @ Sat Oct 11 00:26:11 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:26:12 2025 -------------------
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662394097f1dc/fastq, /tmp/RtmpW5VxCf/file2662394097f1dc/fastq/sample1.fq.gz, /tmp/RtmpW5VxCf/file2662394097f1dc/fastq/sample2.fq.gz, /tmp/RtmpW5VxCf/file2662394097f1dc/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662394097f1dc/sampleA_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662394097f1dc/sample1_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662394097f1dc/sample2_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662394097f1dc/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662394097f1dc/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662394097f1dc/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662394097f1dc/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662394097f1dc/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpW5VxCf/file2662394097f1dc/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file2662394097f1dc/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpW5VxCf/file2662394097f1dc/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file2662394097f1dc/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpW5VxCf/file2662394097f1dc/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file2662394097f1dc/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpW5VxCf/file2662394097f1dc/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file2662394097f1dc/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Oct 11 00:26:14 2025 ----------
2025-10-11T04:26:14.091757Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:26:14.092282Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662394097f1dc/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T04:26:14.092294Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:26:14.092297Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:26:14.092374Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:26:14.092381Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T04:26:14.103881Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-11T04:26:14.646104Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:26:14.646500Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662394097f1dc/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T04:26:14.646510Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:26:14.646513Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:26:14.646598Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:26:14.646606Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T04:26:15.163829Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:26:15.164328Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662394097f1dc/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T04:26:15.164339Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:26:15.164343Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:26:15.164419Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:26:15.164427Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T04:26:15.705023Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:26:15.705396Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662394097f1dc/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T04:26:15.705407Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:26:15.705411Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:26:15.705495Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:26:15.705504Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662396d8aa9d9/config_file_2515513.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 11 00:26:16 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/RtmpW5VxCf/file2662396d8aa9d9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662396d8aa9d9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662396d8aa9d9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662396d8aa9d9/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/RtmpW5VxCf/file2662396d8aa9d9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662396d8aa9d9/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/RtmpW5VxCf/file2662396d8aa9d9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662396d8aa9d9/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/RtmpW5VxCf/file2662396d8aa9d9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662396d8aa9d9/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 @ Sat Oct 11 00:26:17 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_align2genome.bam
/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_align2genome.bam
/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_align2genome.bam
/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 11 00:26:36 2025 ----------------
00:26:36 Sat Oct 11 2025 quantify genes 
Using BAM(s): '/tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_align2genome.bam', and
'/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_align2genome.bam'
parsing /tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.51gene_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, 397881.16Read/s]
parsing /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.93gene_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, 1292782.64Read/s]
parsing /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1367291.69Read/s]
parsing /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.34gene_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, 751505.77Read/s]
-- Running step: isoform_identification @ Sat Oct 11 00:26:37 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:26:37 2025 -------------------
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662396d8aa9d9/fastq, /tmp/RtmpW5VxCf/file2662396d8aa9d9/fastq/sample1.fq.gz, /tmp/RtmpW5VxCf/file2662396d8aa9d9/fastq/sample2.fq.gz, /tmp/RtmpW5VxCf/file2662396d8aa9d9/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:26:57 2025 ----------
2025-10-11T04:26:57.421808Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:26:57.422313Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662396d8aa9d9/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T04:26:57.422325Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:26:57.422328Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:26:57.422408Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:26:57.422416Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T04:26:57.434177Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-11T04:26:58.017599Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:26:58.018041Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T04:26:58.018049Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:26:58.018052Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:26:58.018147Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:26:58.018158Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T04:26:58.604335Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:26:58.604684Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T04:26:58.604691Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:26:58.604694Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:26:58.604780Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:26:58.604787Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-11T04:26:59.127863Z  INFO oarfish: setting user-provided filter parameters.
2025-10-11T04:26:59.128338Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpW5VxCf/file2662396d8aa9d9/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-11T04:26:59.128350Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-11T04:26:59.128352Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-11T04:26:59.128438Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-11T04:26:59.128446Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file26623923bb5322/config_file_2515513.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 11 00:26:59 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/RtmpW5VxCf/file26623923bb5322/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623923bb5322/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623923bb5322/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file26623923bb5322/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 @ Sat Oct 11 00:27:00 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpW5VxCf/file26623923bb5322/sampleA_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/sample1_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/sample2_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/sample3_matched_reads.fastq.gz -> /tmp/RtmpW5VxCf/file26623923bb5322/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 11 00:27:02 2025 ----------------
00:27:02 Sat Oct 11 2025 quantify genes 
Using BAM(s): '/tmp/RtmpW5VxCf/file26623923bb5322/sampleA_align2genome.bam',
'/tmp/RtmpW5VxCf/file26623923bb5322/sample1_align2genome.bam',
'/tmp/RtmpW5VxCf/file26623923bb5322/sample2_align2genome.bam', and
'/tmp/RtmpW5VxCf/file26623923bb5322/sample3_align2genome.bam'
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.64gene_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, 415475.08Read/s]
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.83gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  7.81gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1405032.83Read/s]
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.97gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1368540.85Read/s]
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.92gene_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, 790244.93Read/s]
-- Running step: isoform_identification @ Sat Oct 11 00:27:03 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:27:03 2025 -------------------
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623923bb5322/fastq, /tmp/RtmpW5VxCf/file26623923bb5322/fastq/sample1.fq.gz, /tmp/RtmpW5VxCf/file26623923bb5322/fastq/sample2.fq.gz, /tmp/RtmpW5VxCf/file26623923bb5322/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623923bb5322/sampleA_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623923bb5322/sample1_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623923bb5322/sample2_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file26623923bb5322/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file26623923bb5322/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623923bb5322/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623923bb5322/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file26623923bb5322/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpW5VxCf/file26623923bb5322/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file26623923bb5322/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/RtmpW5VxCf/file26623923bb5322/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpW5VxCf/file26623923bb5322/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 11 00:27:04 2025 ----------
00:27:04 Sat Oct 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sampleA_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file26623923bb5322/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample1_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file26623923bb5322/sample1_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample2_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file26623923bb5322/sample2_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample3_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file26623923bb5322/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file26623923bb5322/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpW5VxCf/file2662397393b6bc/config_file_2515513.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 11 00:27:07 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/RtmpW5VxCf/file2662397393b6bc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662397393b6bc/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662397393b6bc/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpW5VxCf/file2662397393b6bc/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/RtmpW5VxCf/file2662397393b6bc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662397393b6bc/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/RtmpW5VxCf/file2662397393b6bc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662397393b6bc/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/RtmpW5VxCf/file2662397393b6bc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpW5VxCf/file2662397393b6bc/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 @ Sat Oct 11 00:27:08 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_align2genome.bam
/tmp/RtmpW5VxCf/file2662397393b6bc/sample1_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662397393b6bc/sample1_align2genome.bam
/tmp/RtmpW5VxCf/file2662397393b6bc/sample2_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662397393b6bc/sample2_align2genome.bam
/tmp/RtmpW5VxCf/file2662397393b6bc/sample3_matched_reads.fastq.gz ->/tmp/RtmpW5VxCf/file2662397393b6bc/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 11 00:27:27 2025 ----------------
00:27:27 Sat Oct 11 2025 quantify genes 
Using BAM(s): '/tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662397393b6bc/sample1_align2genome.bam',
'/tmp/RtmpW5VxCf/file2662397393b6bc/sample2_align2genome.bam', and
'/tmp/RtmpW5VxCf/file2662397393b6bc/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.27gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 340292.08Read/s]
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.33gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1233183.58Read/s]
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1240918.34Read/s]
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.63gene_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, 696820.84Read/s]
-- Running step: isoform_identification @ Sat Oct 11 00:27:28 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 11 00:27:28 2025 -------------------
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662397393b6bc/fastq, /tmp/RtmpW5VxCf/file2662397393b6bc/fastq/sample1.fq.gz, /tmp/RtmpW5VxCf/file2662397393b6bc/fastq/sample2.fq.gz, /tmp/RtmpW5VxCf/file2662397393b6bc/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662397393b6bc/sample1_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662397393b6bc/sample2_matched_reads.fastq.gz, /tmp/RtmpW5VxCf/file2662397393b6bc/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662397393b6bc/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662397393b6bc/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpW5VxCf/file2662397393b6bc/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662397393b6bc/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662397393b6bc/sample1_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662397393b6bc/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662397393b6bc/sample2_realign2transcript.bam
/tmp/RtmpW5VxCf/file2662397393b6bc/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpW5VxCf/file2662397393b6bc/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 11 00:27:46 2025 ----------
00:27:46 Sat Oct 11 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662397393b6bc/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample1_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662397393b6bc/sample1_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample2_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662397393b6bc/sample2_realign2transcript.bamdone
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample3_realign2transcript.bam...
parsing /tmp/RtmpW5VxCf/file2662397393b6bc/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpW5VxCf/file2662397393b6bc/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 
1693.635   46.806  757.499