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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 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     25.056  0.735  25.791
find_variants                20.157  1.472  21.018
blaze                         4.309 17.265  12.536
bulk_long_pipeline            2.319 13.370   2.459
sc_long_multisample_pipeline  7.995  6.260   8.203
sc_plot_genotype             10.722  0.703  10.254
MultiSampleSCPipeline        10.100  0.713  11.145
sc_DTU_analysis               7.250  2.312   7.457
plot_isoform_heatmap          7.427  0.320   7.746
create_sce_from_dir           3.553  2.898   3.865
sc_long_pipeline              3.239  2.187   2.967
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

Status: 5 NOTEs
See
  ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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

R version 4.5.1 Patched (2025-08-23 r88802) -- "Great Square Root"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d933749c031/config_file_2723219.json 
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d933749c031/config_file_2723219.json 
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d933749c031/config_file_2723219.json 
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d935c154d91/config_file_2723219.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/RtmpehAsOW/file298d9378ad9914/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/RtmpehAsOW/file298d9370a5925d/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/RtmpehAsOW/file298d9370a5925d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d934562bda1/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d934562bda1/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d934562bda1/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d934562bda1/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/RtmpehAsOW/file298d934c546893/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/RtmpehAsOW/file298d9371268fa4/config_file_2723219.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 Sep 20 00:02:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpehAsOW/file298d9371268fa4/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpehAsOW/file298d9371268fa4/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpehAsOW/file298d9371268fa4/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Sep 20 00:02:58 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[00:03:05] 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.

[00:03:05] 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.

[00:03:05] 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.

[00:03:05] 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.

[00:03:07] 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.

[00:03:07] 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 Sep 20 00:03:25 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpehAsOW/file298d9371268fa4/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpehAsOW/file298d9371268fa4/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpehAsOW/file298d9371268fa4/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sat Sep 20 00:03:26 2025 ----------
2025-09-20T04:03:26.441763Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:03:26.442130Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9371268fa4/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:03:26.442139Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:03:26.442143Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:03:26.442203Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:03:26.442209Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:03:26.443724Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:03:26.443853Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-09-20T04:03:26.443875Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-09-20T04:03:26.443889Z  INFO oarfish::bulk: number of aligned reads : 96
2025-09-20T04:03:26.443891Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-20T04:03:26.444497Z  INFO oarfish: oarfish completed successfully.
2025-09-20T04:03:26.451976Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:03:26.452460Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9371268fa4/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:03:26.452468Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:03:26.452472Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:03:26.452531Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:03:26.452537Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:03:26.454143Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:03:26.454287Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-09-20T04:03:26.454313Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-09-20T04:03:26.454315Z  INFO oarfish::bulk: number of aligned reads : 95
2025-09-20T04:03:26.454317Z  INFO oarfish::bulk: number of unique alignments : 82
2025-09-20T04:03:26.454934Z  INFO oarfish: oarfish completed successfully.
2025-09-20T04:03:26.462049Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:03:26.462391Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9371268fa4/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:03:26.462400Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:03:26.462403Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:03:26.462456Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:03:26.462462Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:03:26.465208Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-09-20T04:03:26.465369Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-09-20T04:03:26.465405Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-09-20T04:03:26.465407Z  INFO oarfish::bulk: number of aligned reads : 179
2025-09-20T04:03:26.465409Z  INFO oarfish::bulk: number of unique alignments : 143
2025-09-20T04:03:26.466085Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d93276c89b5/config_file_2723219.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 Sep 20 00:03:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpehAsOW/file298d93276c89b5/sample1_align2genome.bam
sample2 ->/tmp/RtmpehAsOW/file298d93276c89b5/sample2_align2genome.bam
sample3 ->/tmp/RtmpehAsOW/file298d93276c89b5/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Sep 20 00:03:46 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 Sep 20 00:04:08 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpehAsOW/file298d93276c89b5/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpehAsOW/file298d93276c89b5/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpehAsOW/file298d93276c89b5/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:04:29 2025 ----------
2025-09-20T04:04:29.397257Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:04:29.397620Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d93276c89b5/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:04:29.397641Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:04:29.397645Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:04:29.397730Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:04:29.397737Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:04:29.399183Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:04:29.399310Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-09-20T04:04:29.399331Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-09-20T04:04:29.399333Z  INFO oarfish::bulk: number of aligned reads : 96
2025-09-20T04:04:29.399336Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-20T04:04:29.399954Z  INFO oarfish: oarfish completed successfully.
2025-09-20T04:04:29.407887Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:04:29.408409Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d93276c89b5/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:04:29.408418Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:04:29.408421Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:04:29.408478Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:04:29.408484Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:04:29.410072Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:04:29.410208Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-09-20T04:04:29.410233Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-09-20T04:04:29.410244Z  INFO oarfish::bulk: number of aligned reads : 95
2025-09-20T04:04:29.410246Z  INFO oarfish::bulk: number of unique alignments : 82
2025-09-20T04:04:29.410841Z  INFO oarfish: oarfish completed successfully.
2025-09-20T04:04:29.418215Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:04:29.418671Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d93276c89b5/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:04:29.418680Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:04:29.418683Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:04:29.418755Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:04:29.418764Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:04:29.421452Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-09-20T04:04:29.421623Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-09-20T04:04:29.421655Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-09-20T04:04:29.421658Z  INFO oarfish::bulk: number of aligned reads : 179
2025-09-20T04:04:29.421660Z  INFO oarfish::bulk: number of unique alignments : 143
2025-09-20T04:04:29.422361Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d9360b6c951/config_file_2723219.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 Sep 20 00:04:29 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpehAsOW/file298d9360b6c951/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpehAsOW/file298d9360b6c951/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpehAsOW/file298d9360b6c951/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Sep 20 00:04:30 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 Sep 20 00:04:50 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpehAsOW/file298d9360b6c951/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpehAsOW/file298d9360b6c951/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpehAsOW/file298d9360b6c951/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Sep 20 00:04:50 2025 ----------
00:04:50 Sat Sep 20 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/RtmpehAsOW/file298d9327fad07e/config_file_2723219.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 Sep 20 00:04:52 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpehAsOW/file298d9327fad07e/sample1_align2genome.bam
sample2 ->/tmp/RtmpehAsOW/file298d9327fad07e/sample2_align2genome.bam
sample3 ->/tmp/RtmpehAsOW/file298d9327fad07e/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Sep 20 00:05:12 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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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 Sep 20 00:05:31 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpehAsOW/file298d9327fad07e/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpehAsOW/file298d9327fad07e/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpehAsOW/file298d9327fad07e/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:05:50 2025 ----------
00:05:50 Sat Sep 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpehAsOW/file298d9360b6c951/sample1_realign2transcript.bam', '/tmp/RtmpehAsOW/file298d9360b6c951/sample2_realign2transcript.bam', '/tmp/RtmpehAsOW/file298d9360b6c951/sample3_realign2transcript.bam'] /tmp/RtmpehAsOW/file298d9360b6c951/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/RtmpehAsOW/file298d9379f1edd/config_file_2723219.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 Sep 20 00:05:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpehAsOW/file298d9379f1edd/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpehAsOW/file298d9379f1edd/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpehAsOW/file298d9379f1edd/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Sep 20 00:05:52 2025 -------------
Inputs:  ['/tmp/RtmpehAsOW/file298d9327fad07e/sample1_realign2transcript.bam', '/tmp/RtmpehAsOW/file298d9327fad07e/sample2_realign2transcript.bam', '/tmp/RtmpehAsOW/file298d9327fad07e/sample3_realign2transcript.bam'] /tmp/RtmpehAsOW/file298d9327fad07e/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 Sep 20 00:05:52 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpehAsOW/file298d9379f1edd/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpehAsOW/file298d9379f1edd/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpehAsOW/file298d9379f1edd/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sat Sep 20 00:05:53 2025 ----------
2025-09-20T04:05:53.831912Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:05:53.832507Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9379f1edd/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-09-20T04:05:53.832522Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:05:53.832526Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:05:53.832620Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:05:53.832628Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-20T04:05:53.835308Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:05:53.835475Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-09-20T04:05:53.835500Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-09-20T04:05:53.835503Z  INFO oarfish::bulk: number of aligned reads : 98
2025-09-20T04:05:53.835505Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-20T04:05:53.836177Z  INFO oarfish: oarfish completed successfully.
2025-09-20T04:05:53.844276Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:05:53.844690Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9379f1edd/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-09-20T04:05:53.844698Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:05:53.844702Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:05:53.844799Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:05:53.844811Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-20T04:05:53.847456Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:05:53.847600Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-09-20T04:05:53.847626Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-09-20T04:05:53.847629Z  INFO oarfish::bulk: number of aligned reads : 97
2025-09-20T04:05:53.847631Z  INFO oarfish::bulk: number of unique alignments : 79
2025-09-20T04:05:53.848270Z  INFO oarfish: oarfish completed successfully.
2025-09-20T04:05:53.855574Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:05:53.856044Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9379f1edd/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-09-20T04:05:53.856056Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:05:53.856059Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:05:53.856125Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:05:53.856131Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-20T04:05:53.860492Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:05:53.860660Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-09-20T04:05:53.860687Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-09-20T04:05:53.860690Z  INFO oarfish::bulk: number of aligned reads : 187
2025-09-20T04:05:53.860692Z  INFO oarfish::bulk: number of unique alignments : 140
2025-09-20T04:05:53.861418Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d936da56890/config_file_2723219.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 Sep 20 00:05:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpehAsOW/file298d936da56890/sample1_align2genome.bam
sample2 ->/tmp/RtmpehAsOW/file298d936da56890/sample2_align2genome.bam
sample3 ->/tmp/RtmpehAsOW/file298d936da56890/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Sep 20 00:06:13 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:06:13 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpehAsOW/file298d936da56890/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpehAsOW/file298d936da56890/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpehAsOW/file298d936da56890/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:06:32 2025 ----------
2025-09-20T04:06:32.879496Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:06:32.879932Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d936da56890/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-09-20T04:06:32.879943Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:06:32.879960Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:06:32.880038Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:06:32.880045Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-20T04:06:32.882757Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:06:32.882906Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-09-20T04:06:32.882928Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-09-20T04:06:32.882931Z  INFO oarfish::bulk: number of aligned reads : 98
2025-09-20T04:06:32.882933Z  INFO oarfish::bulk: number of unique alignments : 86
2025-09-20T04:06:32.883550Z  INFO oarfish: oarfish completed successfully.
2025-09-20T04:06:32.894363Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:06:32.894916Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d936da56890/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-09-20T04:06:32.894929Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:06:32.894933Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:06:32.895010Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:06:32.895017Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-20T04:06:32.897867Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:06:32.898036Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-09-20T04:06:32.898063Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-09-20T04:06:32.898065Z  INFO oarfish::bulk: number of aligned reads : 97
2025-09-20T04:06:32.898078Z  INFO oarfish::bulk: number of unique alignments : 79
2025-09-20T04:06:32.898702Z  INFO oarfish: oarfish completed successfully.
2025-09-20T04:06:32.909173Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:06:32.909549Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d936da56890/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-09-20T04:06:32.909558Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:06:32.909562Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:06:32.909642Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:06:32.909649Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-20T04:06:32.914050Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-09-20T04:06:32.914252Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-09-20T04:06:32.914281Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-09-20T04:06:32.914284Z  INFO oarfish::bulk: number of aligned reads : 187
2025-09-20T04:06:32.914287Z  INFO oarfish::bulk: number of unique alignments : 140
2025-09-20T04:06:32.915015Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d936a519dde/config_file_2723219.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 Sep 20 00:06:33 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpehAsOW/file298d936a519dde/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpehAsOW/file298d936a519dde/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpehAsOW/file298d936a519dde/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Sep 20 00:06:34 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:06:34 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpehAsOW/file298d936a519dde/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpehAsOW/file298d936a519dde/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpehAsOW/file298d936a519dde/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Sep 20 00:06:35 2025 ----------
00:06:35 Sat Sep 20 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/RtmpehAsOW/file298d9362fc61f9/config_file_2723219.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 Sep 20 00:06:36 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpehAsOW/file298d9362fc61f9/sample1_align2genome.bam
sample2 ->/tmp/RtmpehAsOW/file298d9362fc61f9/sample2_align2genome.bam
sample3 ->/tmp/RtmpehAsOW/file298d9362fc61f9/sample3_align2genome.bam
-- Running step: isoform_identification @ Sat Sep 20 00:06:54 2025 -------------
Inputs:  ['/tmp/RtmpehAsOW/file298d936a519dde/sample1_realign2transcript.bam', '/tmp/RtmpehAsOW/file298d936a519dde/sample2_realign2transcript.bam', '/tmp/RtmpehAsOW/file298d936a519dde/sample3_realign2transcript.bam'] /tmp/RtmpehAsOW/file298d936a519dde/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 Sep 20 00:06:55 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpehAsOW/file298d9362fc61f9/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpehAsOW/file298d9362fc61f9/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpehAsOW/file298d9362fc61f9/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:07:13 2025 ----------
00:07:13 Sat Sep 20 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/RtmpehAsOW/file298d9376fe3ade/config_file_2723219.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 Sep 20 00:07:14 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/RtmpehAsOW/file298d9376fe3ade/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 Sep 20 00:07:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpehAsOW/file298d9376fe3ade/matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d9376fe3ade/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Sep 20 00:07:15 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 Sep 20 00:07:25 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/RtmpehAsOW/file298d9376fe3ade/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9376fe3ade/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpehAsOW/file298d9376fe3ade/matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d9376fe3ade/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 Sep 20 00:07:26 2025 ----------
2025-09-20T04:07:26.092571Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:07:26.092992Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9376fe3ade/realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:07:26.093003Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:07:26.093007Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:07:26.093061Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:07:26.093068Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:07:26.099300Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d933a5d0689/config_file_2723219.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 Sep 20 00:07:26 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d933a5d0689/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 Sep 20 00:07:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpehAsOW/file298d933a5d0689/matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d933a5d0689/align2genome.bam
-- Running step: isoform_identification @ Sat Sep 20 00:07:44 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 Sep 20 00:07:55 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/RtmpehAsOW/file298d933a5d0689/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d933a5d0689/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpehAsOW/file298d933a5d0689/matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d933a5d0689/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:08:13 2025 ----------
2025-09-20T04:08:13.728475Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:08:13.728898Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d933a5d0689/realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:08:13.728909Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:08:13.728913Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:08:13.728972Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:08:13.728979Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:08:13.735668Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d935d73b8de/config_file_2723219.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 Sep 20 00:08:14 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/RtmpehAsOW/file298d935d73b8de/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 Sep 20 00:08:14 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpehAsOW/file298d935d73b8de/matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d935d73b8de/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 Sep 20 00:08:14 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Sep 20 00:08:25 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/RtmpehAsOW/file298d935d73b8de/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d935d73b8de/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpehAsOW/file298d935d73b8de/matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d935d73b8de/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 Sep 20 00:08:25 2025 ----------
00:08:25 Sat Sep 20 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpehAsOW/file298d9362fc61f9/sample1_realign2transcript.bam', '/tmp/RtmpehAsOW/file298d9362fc61f9/sample2_realign2transcript.bam', '/tmp/RtmpehAsOW/file298d9362fc61f9/sample3_realign2transcript.bam'] /tmp/RtmpehAsOW/file298d9362fc61f9/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/RtmpehAsOW/file298d9322245cd8/config_file_2723219.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 Sep 20 00:08:26 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d9322245cd8/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 Sep 20 00:08:26 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9322245cd8/matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9322245cd8/align2genome.bam
-- Running step: isoform_identification @ Sat Sep 20 00:08:44 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 Sep 20 00:08:55 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/RtmpehAsOW/file298d9322245cd8/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9322245cd8/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9322245cd8/matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9322245cd8/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:09:12 2025 ----------
00:09:12 Sat Sep 20 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/RtmpehAsOW/file298d93de6dabd/config_file_2723219.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 Sep 20 00:09:13 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d93de6dabd/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 Sep 20 00:09:13 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpehAsOW/file298d93de6dabd/matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d93de6dabd/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Sat Sep 20 00:09:13 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 Sep 20 00:09:14 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/RtmpehAsOW/file298d93de6dabd/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d93de6dabd/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpehAsOW/file298d93de6dabd/matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d93de6dabd/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 Sep 20 00:09:14 2025 ----------
2025-09-20T04:09:14.287925Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:09:14.288388Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d93de6dabd/realign2transcript.bam, contains 10 reference sequences.
2025-09-20T04:09:14.288395Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:09:14.288398Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:09:14.288466Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:09:14.288473Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-20T04:09:14.297740Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d932301fb1e/config_file_2723219.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 Sep 20 00:09:15 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/RtmpehAsOW/file298d932301fb1e/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 Sep 20 00:09:15 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpehAsOW/file298d932301fb1e/matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d932301fb1e/align2genome.bam
-- Running step: isoform_identification @ Sat Sep 20 00:09:32 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:09:32 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/RtmpehAsOW/file298d932301fb1e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d932301fb1e/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpehAsOW/file298d932301fb1e/matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d932301fb1e/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:09:49 2025 ----------
2025-09-20T04:09:49.972817Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:09:49.973248Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d932301fb1e/realign2transcript.bam, contains 10 reference sequences.
2025-09-20T04:09:49.973259Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:09:49.973263Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:09:49.973340Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:09:49.973347Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-09-20T04:09:49.983151Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d93609169c2/config_file_2723219.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 Sep 20 00:09:50 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d93609169c2/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 Sep 20 00:09:51 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpehAsOW/file298d93609169c2/matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d93609169c2/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 Sep 20 00:09:51 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:09:51 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d93609169c2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d93609169c2/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpehAsOW/file298d93609169c2/matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d93609169c2/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 Sep 20 00:09:51 2025 ----------
00:09:51 Sat Sep 20 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d936ecde3ff/config_file_2723219.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 Sep 20 00:09:53 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/RtmpehAsOW/file298d936ecde3ff/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 Sep 20 00:09:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpehAsOW/file298d936ecde3ff/matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d936ecde3ff/align2genome.bam
-- Running step: isoform_identification @ Sat Sep 20 00:10:10 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:10:11 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d936ecde3ff/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d936ecde3ff/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpehAsOW/file298d936ecde3ff/matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d936ecde3ff/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:10:28 2025 ----------
00:10:28 Sat Sep 20 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d933dbad4d0/config_file_2723219.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 Sep 20 00:10:30 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d933dbad4d0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d933dbad4d0/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d933dbad4d0/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d933dbad4d0/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/RtmpehAsOW/file298d933dbad4d0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d933dbad4d0/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/RtmpehAsOW/file298d933dbad4d0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d933dbad4d0/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/RtmpehAsOW/file298d933dbad4d0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d933dbad4d0/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 Sep 20 00:10:30 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpehAsOW/file298d933dbad4d0/sample1_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d933dbad4d0/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpehAsOW/file298d933dbad4d0/sample2_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d933dbad4d0/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpehAsOW/file298d933dbad4d0/sample3_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d933dbad4d0/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Sep 20 00:10:32 2025 ----------------
00:10:32 Sat Sep 20 2025 quantify genes 
Using BAM(s): '/tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_align2genome.bam',
'/tmp/RtmpehAsOW/file298d933dbad4d0/sample1_align2genome.bam',
'/tmp/RtmpehAsOW/file298d933dbad4d0/sample2_align2genome.bam', and
'/tmp/RtmpehAsOW/file298d933dbad4d0/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.73gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 398880.10Read/s]
parsing /tmp/RtmpehAsOW/file298d933dbad4d0/sample1_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1343811.35Read/s]
parsing /tmp/RtmpehAsOW/file298d933dbad4d0/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.40gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1279063.19Read/s]
parsing /tmp/RtmpehAsOW/file298d933dbad4d0/sample3_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 742933.26Read/s]
-- Running step: isoform_identification @ Sat Sep 20 00:10:34 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  25%
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  |                                                                            
  |====================================================                  |  75%
  |                                                                            
  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Sep 20 00:10:58 2025 -------------------
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d933dbad4d0/fastq, /tmp/RtmpehAsOW/file298d933dbad4d0/fastq/sample1.fq.gz, /tmp/RtmpehAsOW/file298d933dbad4d0/fastq/sample2.fq.gz, /tmp/RtmpehAsOW/file298d933dbad4d0/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d933dbad4d0/sample1_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d933dbad4d0/sample2_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d933dbad4d0/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d933dbad4d0/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d933dbad4d0/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d933dbad4d0/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpehAsOW/file298d933dbad4d0/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d933dbad4d0/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpehAsOW/file298d933dbad4d0/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d933dbad4d0/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpehAsOW/file298d933dbad4d0/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d933dbad4d0/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Sep 20 00:10:59 2025 ----------
2025-09-20T04:10:59.722639Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:10:59.723139Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d933dbad4d0/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:10:59.723161Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:10:59.723165Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:10:59.723226Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:10:59.723233Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:10:59.729125Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-20T04:11:00.054682Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:11:00.055314Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d933dbad4d0/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:11:00.055325Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:11:00.055329Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:11:00.055389Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:11:00.055395Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:11:00.338837Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:11:00.339289Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d933dbad4d0/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:11:00.339296Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:11:00.339299Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:11:00.339356Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:11:00.339362Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:11:00.619551Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:11:00.619913Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d933dbad4d0/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:11:00.619925Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:11:00.619928Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:11:00.619978Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:11:00.619983Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d9368fe854f/config_file_2723219.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 Sep 20 00:11:01 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/RtmpehAsOW/file298d9368fe854f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9368fe854f/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9368fe854f/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9368fe854f/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/RtmpehAsOW/file298d9368fe854f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9368fe854f/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/RtmpehAsOW/file298d9368fe854f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9368fe854f/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/RtmpehAsOW/file298d9368fe854f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9368fe854f/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 Sep 20 00:11:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9368fe854f/sampleA_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9368fe854f/sampleA_align2genome.bam
/tmp/RtmpehAsOW/file298d9368fe854f/sample1_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9368fe854f/sample1_align2genome.bam
/tmp/RtmpehAsOW/file298d9368fe854f/sample2_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9368fe854f/sample2_align2genome.bam
/tmp/RtmpehAsOW/file298d9368fe854f/sample3_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9368fe854f/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Sep 20 00:11:21 2025 ----------------
00:11:21 Sat Sep 20 2025 quantify genes 
Using BAM(s): '/tmp/RtmpehAsOW/file298d9368fe854f/sampleA_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9368fe854f/sample1_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9368fe854f/sample2_align2genome.bam', and
'/tmp/RtmpehAsOW/file298d9368fe854f/sample3_align2genome.bam'
parsing /tmp/RtmpehAsOW/file298d9368fe854f/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 351434.80Read/s]
parsing /tmp/RtmpehAsOW/file298d9368fe854f/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, 1346918.43Read/s]
parsing /tmp/RtmpehAsOW/file298d9368fe854f/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.29gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1242241.44Read/s]
parsing /tmp/RtmpehAsOW/file298d9368fe854f/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.92gene_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, 646032.90Read/s]
-- Running step: isoform_identification @ Sat Sep 20 00:11:22 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Sep 20 00:11:48 2025 -------------------
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9368fe854f/fastq, /tmp/RtmpehAsOW/file298d9368fe854f/fastq/sample1.fq.gz, /tmp/RtmpehAsOW/file298d9368fe854f/fastq/sample2.fq.gz, /tmp/RtmpehAsOW/file298d9368fe854f/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9368fe854f/sampleA_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9368fe854f/sample1_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9368fe854f/sample2_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9368fe854f/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9368fe854f/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9368fe854f/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9368fe854f/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9368fe854f/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9368fe854f/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9368fe854f/sampleA_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9368fe854f/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9368fe854f/sample1_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9368fe854f/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9368fe854f/sample2_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9368fe854f/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9368fe854f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:12:07 2025 ----------
2025-09-20T04:12:07.801646Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:12:07.802051Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9368fe854f/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:12:07.802064Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:12:07.802068Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:12:07.802134Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:12:07.802140Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:12:07.807980Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-20T04:12:08.186916Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:12:08.187393Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9368fe854f/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:12:08.187404Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:12:08.187407Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:12:08.187475Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:12:08.187482Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:12:08.531327Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:12:08.531876Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9368fe854f/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:12:08.531889Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:12:08.531893Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:12:08.531948Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:12:08.531954Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-09-20T04:12:08.872704Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:12:08.873202Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9368fe854f/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-09-20T04:12:08.873213Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:12:08.873216Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:12:08.873276Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:12:08.873292Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d932c871b92/config_file_2723219.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 Sep 20 00:12:09 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d932c871b92/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d932c871b92/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d932c871b92/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d932c871b92/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 Sep 20 00:12:10 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpehAsOW/file298d932c871b92/sampleA_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/sample1_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/sample2_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/sample3_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d932c871b92/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 Sep 20 00:12:11 2025 ----------------
00:12:11 Sat Sep 20 2025 quantify genes 
Using BAM(s): '/tmp/RtmpehAsOW/file298d932c871b92/sampleA_align2genome.bam',
'/tmp/RtmpehAsOW/file298d932c871b92/sample1_align2genome.bam',
'/tmp/RtmpehAsOW/file298d932c871b92/sample2_align2genome.bam', and
'/tmp/RtmpehAsOW/file298d932c871b92/sample3_align2genome.bam'
parsing /tmp/RtmpehAsOW/file298d932c871b92/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.05gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 415771.61Read/s]
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.79gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1423148.75Read/s]
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.94gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1064436.10Read/s]
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.89gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 682577.79Read/s]
-- Running step: isoform_identification @ Sat Sep 20 00:12:12 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
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  |===================================                                   |  50%
  |                                                                            
  |====================================================                  |  75%
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  |======================================================================| 100%

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Sep 20 00:12:37 2025 -------------------
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d932c871b92/fastq, /tmp/RtmpehAsOW/file298d932c871b92/fastq/sample1.fq.gz, /tmp/RtmpehAsOW/file298d932c871b92/fastq/sample2.fq.gz, /tmp/RtmpehAsOW/file298d932c871b92/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d932c871b92/sampleA_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d932c871b92/sample1_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d932c871b92/sample2_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d932c871b92/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d932c871b92/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d932c871b92/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d932c871b92/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d932c871b92/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpehAsOW/file298d932c871b92/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d932c871b92/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/RtmpehAsOW/file298d932c871b92/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d932c871b92/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 Sep 20 00:12:38 2025 ----------
00:12:38 Sat Sep 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpehAsOW/file298d932c871b92/sampleA_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d932c871b92/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d932c871b92/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample1_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d932c871b92/sample1_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample2_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d932c871b92/sample2_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample3_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d932c871b92/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d932c871b92/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d9325f00632/config_file_2723219.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 Sep 20 00:12:40 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d9325f00632/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9325f00632/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9325f00632/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9325f00632/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/RtmpehAsOW/file298d9325f00632/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9325f00632/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/RtmpehAsOW/file298d9325f00632/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9325f00632/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/RtmpehAsOW/file298d9325f00632/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9325f00632/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 Sep 20 00:12:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9325f00632/sampleA_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9325f00632/sampleA_align2genome.bam
/tmp/RtmpehAsOW/file298d9325f00632/sample1_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9325f00632/sample1_align2genome.bam
/tmp/RtmpehAsOW/file298d9325f00632/sample2_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9325f00632/sample2_align2genome.bam
/tmp/RtmpehAsOW/file298d9325f00632/sample3_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9325f00632/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Sep 20 00:13:01 2025 ----------------
00:13:01 Sat Sep 20 2025 quantify genes 
Using BAM(s): '/tmp/RtmpehAsOW/file298d9325f00632/sampleA_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9325f00632/sample1_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9325f00632/sample2_align2genome.bam', and
'/tmp/RtmpehAsOW/file298d9325f00632/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/RtmpehAsOW/file298d9325f00632/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.22gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 329450.80Read/s]
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.86gene_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, 1137160.83Read/s]
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.93gene_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, 1152914.79Read/s]
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.42gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 738433.80Read/s]
-- Running step: isoform_identification @ Sat Sep 20 00:13:02 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 Sep 20 00:13:27 2025 -------------------
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9325f00632/fastq, /tmp/RtmpehAsOW/file298d9325f00632/fastq/sample1.fq.gz, /tmp/RtmpehAsOW/file298d9325f00632/fastq/sample2.fq.gz, /tmp/RtmpehAsOW/file298d9325f00632/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9325f00632/sampleA_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9325f00632/sample1_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9325f00632/sample2_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9325f00632/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9325f00632/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9325f00632/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9325f00632/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9325f00632/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9325f00632/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9325f00632/sampleA_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9325f00632/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9325f00632/sample1_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9325f00632/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9325f00632/sample2_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9325f00632/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9325f00632/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:13:46 2025 ----------
00:13:46 Sat Sep 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpehAsOW/file298d9325f00632/sampleA_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9325f00632/sampleA_realign2transcript.bamdone
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9325f00632/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample1_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9325f00632/sample1_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample2_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9325f00632/sample2_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample3_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9325f00632/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9325f00632/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d93120f41a2/config_file_2723219.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 Sep 20 00:13:48 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d93120f41a2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d93120f41a2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d93120f41a2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d93120f41a2/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/RtmpehAsOW/file298d93120f41a2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d93120f41a2/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/RtmpehAsOW/file298d93120f41a2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d93120f41a2/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/RtmpehAsOW/file298d93120f41a2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d93120f41a2/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 Sep 20 00:13:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpehAsOW/file298d93120f41a2/sampleA_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d93120f41a2/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpehAsOW/file298d93120f41a2/sample1_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d93120f41a2/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpehAsOW/file298d93120f41a2/sample2_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d93120f41a2/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpehAsOW/file298d93120f41a2/sample3_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d93120f41a2/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Sep 20 00:13:50 2025 ----------------
00:13:50 Sat Sep 20 2025 quantify genes 
Using BAM(s): '/tmp/RtmpehAsOW/file298d93120f41a2/sampleA_align2genome.bam',
'/tmp/RtmpehAsOW/file298d93120f41a2/sample1_align2genome.bam',
'/tmp/RtmpehAsOW/file298d93120f41a2/sample2_align2genome.bam', and
'/tmp/RtmpehAsOW/file298d93120f41a2/sample3_align2genome.bam'
	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/RtmpehAsOW/file298d93120f41a2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.39gene_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, 402123.03Read/s]
parsing /tmp/RtmpehAsOW/file298d93120f41a2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.73gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1305335.49Read/s]
parsing /tmp/RtmpehAsOW/file298d93120f41a2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.53gene_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, 1347957.32Read/s]
parsing /tmp/RtmpehAsOW/file298d93120f41a2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.50gene_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, 788521.58Read/s]
-- Running step: isoform_identification @ Sat Sep 20 00:13:51 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:13:51 2025 -------------------
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d93120f41a2/fastq, /tmp/RtmpehAsOW/file298d93120f41a2/fastq/sample1.fq.gz, /tmp/RtmpehAsOW/file298d93120f41a2/fastq/sample2.fq.gz, /tmp/RtmpehAsOW/file298d93120f41a2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d93120f41a2/sampleA_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d93120f41a2/sample1_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d93120f41a2/sample2_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d93120f41a2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d93120f41a2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d93120f41a2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d93120f41a2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d93120f41a2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpehAsOW/file298d93120f41a2/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d93120f41a2/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpehAsOW/file298d93120f41a2/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d93120f41a2/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpehAsOW/file298d93120f41a2/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d93120f41a2/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpehAsOW/file298d93120f41a2/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d93120f41a2/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Sep 20 00:13:53 2025 ----------
2025-09-20T04:13:53.646411Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:13:53.646814Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d93120f41a2/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-09-20T04:13:53.646826Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:13:53.646829Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:13:53.646913Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:13:53.646921Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-20T04:13:53.659119Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-20T04:13:54.226650Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:13:54.227065Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d93120f41a2/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-09-20T04:13:54.227078Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:13:54.227082Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:13:54.227164Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:13:54.227172Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-20T04:13:54.793590Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:13:54.793999Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d93120f41a2/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-09-20T04:13:54.794010Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:13:54.794014Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:13:54.794094Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:13:54.794102Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-20T04:13:55.349085Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:13:55.349558Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d93120f41a2/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-09-20T04:13:55.349569Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:13:55.349573Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:13:55.349655Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:13:55.349664Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d9335d6b42c/config_file_2723219.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 Sep 20 00:13:56 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/RtmpehAsOW/file298d9335d6b42c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9335d6b42c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9335d6b42c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9335d6b42c/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/RtmpehAsOW/file298d9335d6b42c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9335d6b42c/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/RtmpehAsOW/file298d9335d6b42c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9335d6b42c/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/RtmpehAsOW/file298d9335d6b42c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9335d6b42c/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 Sep 20 00:13:56 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_align2genome.bam
/tmp/RtmpehAsOW/file298d9335d6b42c/sample1_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9335d6b42c/sample1_align2genome.bam
/tmp/RtmpehAsOW/file298d9335d6b42c/sample2_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9335d6b42c/sample2_align2genome.bam
/tmp/RtmpehAsOW/file298d9335d6b42c/sample3_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9335d6b42c/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Sep 20 00:14:17 2025 ----------------
00:14:17 Sat Sep 20 2025 quantify genes 
Using BAM(s): '/tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9335d6b42c/sample1_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9335d6b42c/sample2_align2genome.bam', and
'/tmp/RtmpehAsOW/file298d9335d6b42c/sample3_align2genome.bam'
parsing /tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.33gene_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, 373451.10Read/s]
parsing /tmp/RtmpehAsOW/file298d9335d6b42c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1181494.08Read/s]
parsing /tmp/RtmpehAsOW/file298d9335d6b42c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1286596.32Read/s]
parsing /tmp/RtmpehAsOW/file298d9335d6b42c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.33gene_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, 753016.88Read/s]
-- Running step: isoform_identification @ Sat Sep 20 00:14:18 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:14:18 2025 -------------------
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9335d6b42c/fastq, /tmp/RtmpehAsOW/file298d9335d6b42c/fastq/sample1.fq.gz, /tmp/RtmpehAsOW/file298d9335d6b42c/fastq/sample2.fq.gz, /tmp/RtmpehAsOW/file298d9335d6b42c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9335d6b42c/sample1_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9335d6b42c/sample2_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9335d6b42c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9335d6b42c/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9335d6b42c/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9335d6b42c/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9335d6b42c/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9335d6b42c/sample1_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9335d6b42c/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9335d6b42c/sample2_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9335d6b42c/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9335d6b42c/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:14:38 2025 ----------
2025-09-20T04:14:38.197084Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:14:38.197475Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9335d6b42c/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-09-20T04:14:38.197483Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:14:38.197486Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:14:38.197563Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:14:38.197571Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-20T04:14:38.209576Z  INFO oarfish::single_cell: Processed 100 cells.
2025-09-20T04:14:38.863208Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:14:38.863541Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9335d6b42c/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-09-20T04:14:38.863549Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:14:38.863552Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:14:38.863629Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:14:38.863636Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-20T04:14:39.448433Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:14:39.448920Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9335d6b42c/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-09-20T04:14:39.448932Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:14:39.448935Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:14:39.449014Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:14:39.449021Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-09-20T04:14:39.977909Z  INFO oarfish: setting user-provided filter parameters.
2025-09-20T04:14:39.978430Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpehAsOW/file298d9335d6b42c/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-09-20T04:14:39.978439Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-09-20T04:14:39.978442Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-09-20T04:14:39.978530Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-09-20T04:14:39.978537Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d9358fc9e6c/config_file_2723219.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 Sep 20 00:14:40 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 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d9358fc9e6c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9358fc9e6c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9358fc9e6c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9358fc9e6c/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 Sep 20 00:14:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/sample1_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/sample2_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/sample3_matched_reads.fastq.gz -> /tmp/RtmpehAsOW/file298d9358fc9e6c/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 Sep 20 00:14:42 2025 ----------------
00:14:43 Sat Sep 20 2025 quantify genes 
Using BAM(s): '/tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9358fc9e6c/sample1_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9358fc9e6c/sample2_align2genome.bam', and
'/tmp/RtmpehAsOW/file298d9358fc9e6c/sample3_align2genome.bam'
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 418392.79Read/s]
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  9.08gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  9.06gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1387555.91Read/s]
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.38gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1226691.62Read/s]
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.04gene_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, 691672.82Read/s]
-- Running step: isoform_identification @ Sat Sep 20 00:14:43 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:14:44 2025 -------------------
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9358fc9e6c/fastq, /tmp/RtmpehAsOW/file298d9358fc9e6c/fastq/sample1.fq.gz, /tmp/RtmpehAsOW/file298d9358fc9e6c/fastq/sample2.fq.gz, /tmp/RtmpehAsOW/file298d9358fc9e6c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9358fc9e6c/sample1_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9358fc9e6c/sample2_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9358fc9e6c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9358fc9e6c/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9358fc9e6c/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9358fc9e6c/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d9358fc9e6c/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/RtmpehAsOW/file298d9358fc9e6c/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpehAsOW/file298d9358fc9e6c/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 Sep 20 00:14:45 2025 ----------
00:14:45 Sat Sep 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9358fc9e6c/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample1_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9358fc9e6c/sample1_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample2_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9358fc9e6c/sample2_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample3_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9358fc9e6c/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9358fc9e6c/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpehAsOW/file298d9349442417/config_file_2723219.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 Sep 20 00:14:48 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpehAsOW/file298d9349442417/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9349442417/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9349442417/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpehAsOW/file298d9349442417/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/RtmpehAsOW/file298d9349442417/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9349442417/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/RtmpehAsOW/file298d9349442417/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9349442417/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/RtmpehAsOW/file298d9349442417/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpehAsOW/file298d9349442417/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 Sep 20 00:14:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9349442417/sampleA_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9349442417/sampleA_align2genome.bam
/tmp/RtmpehAsOW/file298d9349442417/sample1_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9349442417/sample1_align2genome.bam
/tmp/RtmpehAsOW/file298d9349442417/sample2_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9349442417/sample2_align2genome.bam
/tmp/RtmpehAsOW/file298d9349442417/sample3_matched_reads.fastq.gz ->/tmp/RtmpehAsOW/file298d9349442417/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Sep 20 00:15:07 2025 ----------------
00:15:07 Sat Sep 20 2025 quantify genes 
Using BAM(s): '/tmp/RtmpehAsOW/file298d9349442417/sampleA_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9349442417/sample1_align2genome.bam',
'/tmp/RtmpehAsOW/file298d9349442417/sample2_align2genome.bam', and
'/tmp/RtmpehAsOW/file298d9349442417/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpehAsOW/file298d9349442417/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.20gene_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, 324355.36Read/s]
parsing /tmp/RtmpehAsOW/file298d9349442417/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.68gene_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, 1344500.58Read/s]
parsing /tmp/RtmpehAsOW/file298d9349442417/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1256983.94Read/s]
parsing /tmp/RtmpehAsOW/file298d9349442417/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.03gene_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, 729190.54Read/s]
-- Running step: isoform_identification @ Sat Sep 20 00:15:08 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Sep 20 00:15:09 2025 -------------------
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9349442417/fastq, /tmp/RtmpehAsOW/file298d9349442417/fastq/sample1.fq.gz, /tmp/RtmpehAsOW/file298d9349442417/fastq/sample2.fq.gz, /tmp/RtmpehAsOW/file298d9349442417/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9349442417/sampleA_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9349442417/sample1_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9349442417/sample2_matched_reads.fastq.gz, /tmp/RtmpehAsOW/file298d9349442417/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpehAsOW/file298d9349442417/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9349442417/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9349442417/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpehAsOW/file298d9349442417/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpehAsOW/file298d9349442417/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9349442417/sampleA_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9349442417/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9349442417/sample1_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9349442417/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9349442417/sample2_realign2transcript.bam
/tmp/RtmpehAsOW/file298d9349442417/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpehAsOW/file298d9349442417/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Sep 20 00:15:27 2025 ----------
00:15:27 Sat Sep 20 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpehAsOW/file298d9349442417/sampleA_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9349442417/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9349442417/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9349442417/sample1_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9349442417/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9349442417/sample1_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9349442417/sample2_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9349442417/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9349442417/sample2_realign2transcript.bamdone
parsing /tmp/RtmpehAsOW/file298d9349442417/sample3_realign2transcript.bam...
parsing /tmp/RtmpehAsOW/file298d9349442417/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpehAsOW/file298d9349442417/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 
1721.985   48.936  772.226