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###
### Running command:
###
###   /home/biocbuild/bbs-3.23-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.23-bioc/R/site-library --timings FLAMES_2.5.2.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck’
* using R Under development (unstable) (2026-01-15 r89304)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.5.2’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... INFO
  specified C++17
* checking installed package size ... OK
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
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 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 ‘outdir’
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 global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     24.398  0.578  24.979
find_variants                21.990  1.110  22.512
blaze                         5.045 17.562  13.473
bulk_long_pipeline            2.355 13.438   2.574
sc_long_multisample_pipeline  8.149  6.338   8.105
sc_plot_genotype             11.772  0.202  10.754
MultiSampleSCPipeline        10.699  1.068  12.226
sc_DTU_analysis               7.057  2.108   6.913
plot_isoform_heatmap          7.229  0.234   7.462
create_sce_from_dir           3.567  2.490   3.792
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

Status: 5 NOTEs
See
  ‘/home/biocbuild/bbs-3.23-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.23-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
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* installing to library ‘/home/biocbuild/bbs-3.23-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.5.2’
** using non-staged installation via StagedInstall field
** libs
specified C++17
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.23-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.23-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.23-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.23-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.23-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.23-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R Under development (unstable) (2026-01-15 r89304) -- "Unsuffered Consequences"
Copyright (C) 2026 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

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

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Type 'contributors()' for more information and
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> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d13c94f780/config_file_3838673.json 
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d13c94f780/config_file_3838673.json 
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d13c94f780/config_file_3838673.json 
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d120f3507f/config_file_3838673.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/RtmpP10EfK/file3a92d148a8913a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpP10EfK/file3a92d11be15429/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpP10EfK/file3a92d11be15429/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d13cf6a364/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d13cf6a364/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d13cf6a364/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d13cf6a364/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/RtmpP10EfK/file3a92d1ae4d94b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d17a9115e4/config_file_3838673.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 @ Wed Feb 25 00:09:01 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpP10EfK/file3a92d17a9115e4/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpP10EfK/file3a92d17a9115e4/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpP10EfK/file3a92d17a9115e4/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Feb 25 00:09:02 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Feb 25 00:09:29 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpP10EfK/file3a92d17a9115e4/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpP10EfK/file3a92d17a9115e4/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpP10EfK/file3a92d17a9115e4/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Feb 25 00:09:29 2026 ----------
2026-02-25T05:09:29.697392Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:09:29.697826Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d17a9115e4/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:09:29.697847Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:09:29.697886Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:09:29.697955Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:09:29.697966Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:09:29.699459Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:09:29.699648Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-25T05:09:29.699688Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-02-25T05:09:29.699695Z  INFO oarfish::bulk: number of aligned reads : 96
2026-02-25T05:09:29.699702Z  INFO oarfish::bulk: number of unique alignments : 86
2026-02-25T05:09:29.700417Z  INFO oarfish: oarfish completed successfully.
2026-02-25T05:09:29.708722Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:09:29.709155Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d17a9115e4/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:09:29.709176Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:09:29.709184Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:09:29.709258Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:09:29.709269Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:09:29.710797Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:09:29.710943Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-25T05:09:29.710990Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-02-25T05:09:29.710997Z  INFO oarfish::bulk: number of aligned reads : 95
2026-02-25T05:09:29.711012Z  INFO oarfish::bulk: number of unique alignments : 82
2026-02-25T05:09:29.711653Z  INFO oarfish: oarfish completed successfully.
2026-02-25T05:09:29.719871Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:09:29.720267Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d17a9115e4/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:09:29.720315Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:09:29.720323Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:09:29.720442Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:09:29.720461Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:09:29.723052Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-02-25T05:09:29.723244Z  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   │
╰─────────────────────────────────┴───────╯

2026-02-25T05:09:29.723300Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-02-25T05:09:29.723307Z  INFO oarfish::bulk: number of aligned reads : 179
2026-02-25T05:09:29.723313Z  INFO oarfish::bulk: number of unique alignments : 143
2026-02-25T05:09:29.724083Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d17c311c82/config_file_3838673.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 @ Wed Feb 25 00:09:29 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpP10EfK/file3a92d17c311c82/sample1_align2genome.bam
sample2 ->/tmp/RtmpP10EfK/file3a92d17c311c82/sample2_align2genome.bam
sample3 ->/tmp/RtmpP10EfK/file3a92d17c311c82/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Feb 25 00:09:53 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Feb 25 00:10:13 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpP10EfK/file3a92d17c311c82/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpP10EfK/file3a92d17c311c82/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpP10EfK/file3a92d17c311c82/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:10:33 2026 ----------
2026-02-25T05:10:33.917222Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:10:33.917768Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d17c311c82/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:10:33.917820Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:10:33.917829Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:10:33.917888Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:10:33.917905Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:10:33.919894Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:10:33.920053Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-25T05:10:33.920098Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-02-25T05:10:33.920106Z  INFO oarfish::bulk: number of aligned reads : 96
2026-02-25T05:10:33.920112Z  INFO oarfish::bulk: number of unique alignments : 86
2026-02-25T05:10:33.920815Z  INFO oarfish: oarfish completed successfully.
2026-02-25T05:10:33.929844Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:10:33.930314Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d17c311c82/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:10:33.930334Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:10:33.930382Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:10:33.930444Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:10:33.930455Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:10:33.932014Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:10:33.932189Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-25T05:10:33.932235Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-02-25T05:10:33.932243Z  INFO oarfish::bulk: number of aligned reads : 95
2026-02-25T05:10:33.932249Z  INFO oarfish::bulk: number of unique alignments : 82
2026-02-25T05:10:33.932945Z  INFO oarfish: oarfish completed successfully.
2026-02-25T05:10:33.941877Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:10:33.942385Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d17c311c82/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:10:33.942407Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:10:33.942415Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:10:33.942508Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:10:33.942519Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:10:33.945245Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-02-25T05:10:33.945478Z  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   │
╰─────────────────────────────────┴───────╯

2026-02-25T05:10:33.945545Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-02-25T05:10:33.945553Z  INFO oarfish::bulk: number of aligned reads : 179
2026-02-25T05:10:33.945574Z  INFO oarfish::bulk: number of unique alignments : 143
2026-02-25T05:10:33.946332Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d1674e133b/config_file_3838673.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 @ Wed Feb 25 00:10:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpP10EfK/file3a92d1674e133b/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpP10EfK/file3a92d1674e133b/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpP10EfK/file3a92d1674e133b/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Feb 25 00:10:35 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |=======================                                               |  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 @ Wed Feb 25 00:10:56 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpP10EfK/file3a92d1674e133b/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpP10EfK/file3a92d1674e133b/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpP10EfK/file3a92d1674e133b/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Feb 25 00:10:57 2026 ----------
00:10:57 Wed Feb 25 2026 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/RtmpP10EfK/file3a92d14712f83/config_file_3838673.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 @ Wed Feb 25 00:10:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpP10EfK/file3a92d14712f83/sample1_align2genome.bam
sample2 ->/tmp/RtmpP10EfK/file3a92d14712f83/sample2_align2genome.bam
sample3 ->/tmp/RtmpP10EfK/file3a92d14712f83/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Feb 25 00:11:19 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Feb 25 00:11:37 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpP10EfK/file3a92d14712f83/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpP10EfK/file3a92d14712f83/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpP10EfK/file3a92d14712f83/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:11:57 2026 ----------
00:11:57 Wed Feb 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpP10EfK/file3a92d1674e133b/sample1_realign2transcript.bam', '/tmp/RtmpP10EfK/file3a92d1674e133b/sample2_realign2transcript.bam', '/tmp/RtmpP10EfK/file3a92d1674e133b/sample3_realign2transcript.bam'] /tmp/RtmpP10EfK/file3a92d1674e133b/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/RtmpP10EfK/file3a92d14e3741ee/config_file_3838673.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 @ Wed Feb 25 00:11:58 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpP10EfK/file3a92d14e3741ee/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpP10EfK/file3a92d14e3741ee/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpP10EfK/file3a92d14e3741ee/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Feb 25 00:11:58 2026 -------------
Inputs:  ['/tmp/RtmpP10EfK/file3a92d14712f83/sample1_realign2transcript.bam', '/tmp/RtmpP10EfK/file3a92d14712f83/sample2_realign2transcript.bam', '/tmp/RtmpP10EfK/file3a92d14712f83/sample3_realign2transcript.bam'] /tmp/RtmpP10EfK/file3a92d14712f83/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 @ Wed Feb 25 00:11:59 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpP10EfK/file3a92d14e3741ee/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpP10EfK/file3a92d14e3741ee/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpP10EfK/file3a92d14e3741ee/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Feb 25 00:12:00 2026 ----------
2026-02-25T05:12:00.303609Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:12:00.304030Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d14e3741ee/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-02-25T05:12:00.304087Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:12:00.304095Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:12:00.304171Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:12:00.304184Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-25T05:12:00.306881Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:12:00.307063Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-25T05:12:00.307123Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-02-25T05:12:00.307131Z  INFO oarfish::bulk: number of aligned reads : 98
2026-02-25T05:12:00.307138Z  INFO oarfish::bulk: number of unique alignments : 86
2026-02-25T05:12:00.307823Z  INFO oarfish: oarfish completed successfully.
2026-02-25T05:12:00.315891Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:12:00.316357Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d14e3741ee/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-02-25T05:12:00.316390Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:12:00.316420Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:12:00.316494Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:12:00.316506Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-25T05:12:00.319183Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:12:00.319356Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-25T05:12:00.319436Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-02-25T05:12:00.319443Z  INFO oarfish::bulk: number of aligned reads : 97
2026-02-25T05:12:00.319449Z  INFO oarfish::bulk: number of unique alignments : 79
2026-02-25T05:12:00.320075Z  INFO oarfish: oarfish completed successfully.
2026-02-25T05:12:00.327827Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:12:00.328233Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d14e3741ee/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-02-25T05:12:00.328258Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:12:00.328265Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:12:00.328350Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:12:00.328363Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-25T05:12:00.332815Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:12:00.332979Z  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   │
╰─────────────────────────────────┴───────╯

2026-02-25T05:12:00.333031Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-02-25T05:12:00.333039Z  INFO oarfish::bulk: number of aligned reads : 187
2026-02-25T05:12:00.333051Z  INFO oarfish::bulk: number of unique alignments : 140
2026-02-25T05:12:00.333824Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d1e721e49/config_file_3838673.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 @ Wed Feb 25 00:12:00 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpP10EfK/file3a92d1e721e49/sample1_align2genome.bam
sample2 ->/tmp/RtmpP10EfK/file3a92d1e721e49/sample2_align2genome.bam
sample3 ->/tmp/RtmpP10EfK/file3a92d1e721e49/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Feb 25 00:12:21 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:12:21 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpP10EfK/file3a92d1e721e49/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpP10EfK/file3a92d1e721e49/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpP10EfK/file3a92d1e721e49/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:12:41 2026 ----------
2026-02-25T05:12:41.822387Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:12:41.822864Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d1e721e49/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-02-25T05:12:41.822885Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:12:41.822928Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:12:41.823003Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:12:41.823014Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-25T05:12:41.825646Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:12:41.825794Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-25T05:12:41.825835Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-02-25T05:12:41.825842Z  INFO oarfish::bulk: number of aligned reads : 98
2026-02-25T05:12:41.825848Z  INFO oarfish::bulk: number of unique alignments : 86
2026-02-25T05:12:41.826474Z  INFO oarfish: oarfish completed successfully.
2026-02-25T05:12:41.835479Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:12:41.835871Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d1e721e49/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-02-25T05:12:41.835891Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:12:41.835898Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:12:41.835978Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:12:41.835991Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-25T05:12:41.838705Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:12:41.838835Z  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    │
╰─────────────────────────────────┴───────╯

2026-02-25T05:12:41.838886Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-02-25T05:12:41.838894Z  INFO oarfish::bulk: number of aligned reads : 97
2026-02-25T05:12:41.838907Z  INFO oarfish::bulk: number of unique alignments : 79
2026-02-25T05:12:41.839556Z  INFO oarfish: oarfish completed successfully.
2026-02-25T05:12:41.848634Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:12:41.848987Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d1e721e49/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-02-25T05:12:41.849031Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:12:41.849039Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:12:41.849106Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:12:41.849126Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-25T05:12:41.853558Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-02-25T05:12:41.853721Z  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   │
╰─────────────────────────────────┴───────╯

2026-02-25T05:12:41.853775Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-02-25T05:12:41.853783Z  INFO oarfish::bulk: number of aligned reads : 187
2026-02-25T05:12:41.853789Z  INFO oarfish::bulk: number of unique alignments : 140
2026-02-25T05:12:41.854526Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d137f1f10c/config_file_3838673.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 @ Wed Feb 25 00:12:42 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpP10EfK/file3a92d137f1f10c/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpP10EfK/file3a92d137f1f10c/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpP10EfK/file3a92d137f1f10c/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Feb 25 00:12:42 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:12:43 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpP10EfK/file3a92d137f1f10c/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpP10EfK/file3a92d137f1f10c/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpP10EfK/file3a92d137f1f10c/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Feb 25 00:12:44 2026 ----------
00:12:44 Wed Feb 25 2026 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/RtmpP10EfK/file3a92d1677e038b/config_file_3838673.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 @ Wed Feb 25 00:12:44 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpP10EfK/file3a92d1677e038b/sample1_align2genome.bam
sample2 ->/tmp/RtmpP10EfK/file3a92d1677e038b/sample2_align2genome.bam
sample3 ->/tmp/RtmpP10EfK/file3a92d1677e038b/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Feb 25 00:13:03 2026 -------------
Inputs:  ['/tmp/RtmpP10EfK/file3a92d137f1f10c/sample1_realign2transcript.bam', '/tmp/RtmpP10EfK/file3a92d137f1f10c/sample2_realign2transcript.bam', '/tmp/RtmpP10EfK/file3a92d137f1f10c/sample3_realign2transcript.bam'] /tmp/RtmpP10EfK/file3a92d137f1f10c/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 @ Wed Feb 25 00:13:04 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpP10EfK/file3a92d1677e038b/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpP10EfK/file3a92d1677e038b/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpP10EfK/file3a92d1677e038b/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:13:23 2026 ----------
00:13:23 Wed Feb 25 2026 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/RtmpP10EfK/file3a92d138ab3ba/config_file_3838673.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 @ Wed Feb 25 00:13:24 2026 ----------------
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/RtmpP10EfK/file3a92d138ab3ba/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Feb 25 00:13:24 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpP10EfK/file3a92d138ab3ba/matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d138ab3ba/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Feb 25 00:13:25 2026 -------------
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 @ Wed Feb 25 00:13:34 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d138ab3ba/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d138ab3ba/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpP10EfK/file3a92d138ab3ba/matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d138ab3ba/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 @ Wed Feb 25 00:13:34 2026 ----------
2026-02-25T05:13:34.378627Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:13:34.379109Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d138ab3ba/realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:13:34.379131Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:13:34.379171Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:13:34.379225Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:13:34.379237Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:13:34.385584Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d13a88e895/config_file_3838673.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 @ Wed Feb 25 00:13:34 2026 ----------------
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/RtmpP10EfK/file3a92d13a88e895/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Feb 25 00:13:35 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d13a88e895/matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d13a88e895/align2genome.bam
-- Running step: isoform_identification @ Wed Feb 25 00:13:53 2026 -------------
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 @ Wed Feb 25 00:14:02 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d13a88e895/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d13a88e895/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d13a88e895/matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d13a88e895/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:14:22 2026 ----------
2026-02-25T05:14:22.335507Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:14:22.335957Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d13a88e895/realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:14:22.335982Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:14:22.336030Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:14:22.336089Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:14:22.336100Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:14:22.343692Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d116e128aa/config_file_3838673.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 @ Wed Feb 25 00:14:22 2026 ----------------
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/RtmpP10EfK/file3a92d116e128aa/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Feb 25 00:14:23 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpP10EfK/file3a92d116e128aa/matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d116e128aa/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 @ Wed Feb 25 00:14:23 2026 -------------
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 @ Wed Feb 25 00:14:33 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d116e128aa/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d116e128aa/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpP10EfK/file3a92d116e128aa/matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d116e128aa/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 @ Wed Feb 25 00:14:33 2026 ----------
00:14:33 Wed Feb 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpP10EfK/file3a92d1677e038b/sample1_realign2transcript.bam', '/tmp/RtmpP10EfK/file3a92d1677e038b/sample2_realign2transcript.bam', '/tmp/RtmpP10EfK/file3a92d1677e038b/sample3_realign2transcript.bam'] /tmp/RtmpP10EfK/file3a92d1677e038b/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/RtmpP10EfK/file3a92d118f347c0/config_file_3838673.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 @ Wed Feb 25 00:14:34 2026 ----------------
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/RtmpP10EfK/file3a92d118f347c0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Feb 25 00:14:34 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d118f347c0/matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d118f347c0/align2genome.bam
-- Running step: isoform_identification @ Wed Feb 25 00:14:53 2026 -------------
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 @ Wed Feb 25 00:15:03 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d118f347c0/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d118f347c0/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d118f347c0/matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d118f347c0/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:15:22 2026 ----------
00:15:22 Wed Feb 25 2026 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/RtmpP10EfK/file3a92d17ae9c0cd/config_file_3838673.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 @ Wed Feb 25 00:15:23 2026 ----------------
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/RtmpP10EfK/file3a92d17ae9c0cd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Feb 25 00:15:23 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpP10EfK/file3a92d17ae9c0cd/matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d17ae9c0cd/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Feb 25 00:15:23 2026 -------------
	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 @ Wed Feb 25 00:15:24 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d17ae9c0cd/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d17ae9c0cd/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpP10EfK/file3a92d17ae9c0cd/matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d17ae9c0cd/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 @ Wed Feb 25 00:15:24 2026 ----------
2026-02-25T05:15:24.565174Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:15:24.565658Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d17ae9c0cd/realign2transcript.bam, contains 10 reference sequences.
2026-02-25T05:15:24.565713Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:15:24.565721Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:15:24.565786Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:15:24.565798Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-25T05:15:24.575457Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d15a66a21c/config_file_3838673.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 @ Wed Feb 25 00:15:25 2026 ----------------
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/RtmpP10EfK/file3a92d15a66a21c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Feb 25 00:15:25 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d15a66a21c/matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d15a66a21c/align2genome.bam
-- Running step: isoform_identification @ Wed Feb 25 00:15:44 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:15:44 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d15a66a21c/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d15a66a21c/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d15a66a21c/matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d15a66a21c/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:16:07 2026 ----------
2026-02-25T05:16:07.502034Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:16:07.502628Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d15a66a21c/realign2transcript.bam, contains 10 reference sequences.
2026-02-25T05:16:07.502650Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:16:07.502657Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:16:07.502763Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:16:07.502777Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-02-25T05:16:07.512772Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d165af5394/config_file_3838673.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 @ Wed Feb 25 00:16:08 2026 ----------------
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/RtmpP10EfK/file3a92d165af5394/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Feb 25 00:16:08 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpP10EfK/file3a92d165af5394/matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d165af5394/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 @ Wed Feb 25 00:16:08 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:16:09 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d165af5394/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d165af5394/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpP10EfK/file3a92d165af5394/matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d165af5394/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 @ Wed Feb 25 00:16:09 2026 ----------
00:16:09 Wed Feb 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d15ad15a67/config_file_3838673.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 @ Wed Feb 25 00:16:10 2026 ----------------
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/RtmpP10EfK/file3a92d15ad15a67/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Wed Feb 25 00:16:10 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d15ad15a67/matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d15ad15a67/align2genome.bam
-- Running step: isoform_identification @ Wed Feb 25 00:16:29 2026 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:16:29 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d15ad15a67/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d15ad15a67/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d15ad15a67/matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d15ad15a67/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:16:48 2026 ----------
00:16:48 Wed Feb 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d129a33ccf/config_file_3838673.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 @ Wed Feb 25 00:16:49 2026 ----------------
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/RtmpP10EfK/file3a92d129a33ccf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d129a33ccf/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d129a33ccf/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d129a33ccf/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/RtmpP10EfK/file3a92d129a33ccf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d129a33ccf/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/RtmpP10EfK/file3a92d129a33ccf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d129a33ccf/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/RtmpP10EfK/file3a92d129a33ccf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d129a33ccf/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 @ Wed Feb 25 00:16:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Feb 25 00:16:51 2026 ----------------
00:16:51 Wed Feb 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_align2genome.bam', and
'/tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.17gene_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, 433349.59Read/s]
parsing /tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.48gene_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, 1393456.48Read/s]
parsing /tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.61gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1349866.12Read/s]
parsing /tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.44gene_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, 721513.80Read/s]
-- Running step: isoform_identification @ Wed Feb 25 00:16:53 2026 -------------
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 @ Wed Feb 25 00:17:16 2026 -------------------
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d129a33ccf/fastq, /tmp/RtmpP10EfK/file3a92d129a33ccf/fastq/sample1.fq.gz, /tmp/RtmpP10EfK/file3a92d129a33ccf/fastq/sample2.fq.gz, /tmp/RtmpP10EfK/file3a92d129a33ccf/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Feb 25 00:17:17 2026 ----------
2026-02-25T05:17:17.268063Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:17:17.268702Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d129a33ccf/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:17:17.268768Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:17:17.268777Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:17:17.268832Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:17:17.268855Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:17:17.274603Z  INFO oarfish::single_cell: Processed 100 cells.
2026-02-25T05:17:17.557868Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:17:17.558277Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d129a33ccf/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:17:17.558351Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:17:17.558359Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:17:17.558429Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:17:17.558453Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:17:17.848967Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:17:17.849437Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d129a33ccf/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:17:17.849456Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:17:17.849502Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:17:17.849557Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:17:17.849568Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:17:18.155930Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:17:18.156360Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d129a33ccf/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:17:18.156390Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:17:18.156398Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:17:18.156474Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:17:18.156485Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d137412d58/config_file_3838673.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 @ Wed Feb 25 00:17:18 2026 ----------------
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/RtmpP10EfK/file3a92d137412d58/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d137412d58/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d137412d58/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d137412d58/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/RtmpP10EfK/file3a92d137412d58/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d137412d58/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/RtmpP10EfK/file3a92d137412d58/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d137412d58/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/RtmpP10EfK/file3a92d137412d58/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d137412d58/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 @ Wed Feb 25 00:17:19 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d137412d58/sampleA_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d137412d58/sampleA_align2genome.bam
/tmp/RtmpP10EfK/file3a92d137412d58/sample1_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d137412d58/sample1_align2genome.bam
/tmp/RtmpP10EfK/file3a92d137412d58/sample2_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d137412d58/sample2_align2genome.bam
/tmp/RtmpP10EfK/file3a92d137412d58/sample3_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d137412d58/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Feb 25 00:17:40 2026 ----------------
00:17:40 Wed Feb 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpP10EfK/file3a92d137412d58/sampleA_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d137412d58/sample1_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d137412d58/sample2_align2genome.bam', and
'/tmp/RtmpP10EfK/file3a92d137412d58/sample3_align2genome.bam'
parsing /tmp/RtmpP10EfK/file3a92d137412d58/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.45gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 394053.36Read/s]
parsing /tmp/RtmpP10EfK/file3a92d137412d58/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1386638.46Read/s]
parsing /tmp/RtmpP10EfK/file3a92d137412d58/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.81gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1335935.79Read/s]
parsing /tmp/RtmpP10EfK/file3a92d137412d58/sample3_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 780538.93Read/s]
-- Running step: isoform_identification @ Wed Feb 25 00:17:41 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Feb 25 00:18:05 2026 -------------------
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d137412d58/fastq, /tmp/RtmpP10EfK/file3a92d137412d58/fastq/sample1.fq.gz, /tmp/RtmpP10EfK/file3a92d137412d58/fastq/sample2.fq.gz, /tmp/RtmpP10EfK/file3a92d137412d58/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d137412d58/sampleA_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d137412d58/sample1_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d137412d58/sample2_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d137412d58/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d137412d58/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d137412d58/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d137412d58/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d137412d58/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d137412d58/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d137412d58/sampleA_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d137412d58/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d137412d58/sample1_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d137412d58/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d137412d58/sample2_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d137412d58/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d137412d58/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:18:24 2026 ----------
2026-02-25T05:18:24.841406Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:18:24.841779Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d137412d58/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:18:24.841844Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:18:24.841851Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:18:24.841901Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:18:24.841911Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:18:24.847543Z  INFO oarfish::single_cell: Processed 100 cells.
2026-02-25T05:18:25.196974Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:18:25.197344Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d137412d58/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:18:25.197407Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:18:25.197415Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:18:25.197467Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:18:25.197478Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:18:25.523417Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:18:25.523933Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d137412d58/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:18:25.523951Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:18:25.523959Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:18:25.524054Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:18:25.524065Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-02-25T05:18:25.826978Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:18:25.827482Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d137412d58/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-02-25T05:18:25.827559Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:18:25.827567Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:18:25.827622Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:18:25.827645Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d15ed4b3d3/config_file_3838673.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 @ Wed Feb 25 00:18:26 2026 ----------------
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/RtmpP10EfK/file3a92d15ed4b3d3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d15ed4b3d3/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d15ed4b3d3/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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 @ Wed Feb 25 00:18:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/sample1_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/sample2_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/sample3_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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 @ Wed Feb 25 00:18:28 2026 ----------------
00:18:28 Wed Feb 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample1_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample2_align2genome.bam', and
'/tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample3_align2genome.bam'
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 466759.85Read/s]
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.92gene_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, 1279063.19Read/s]
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.46gene_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, 1255930.05Read/s]
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.21gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 782694.63Read/s]
-- Running step: isoform_identification @ Wed Feb 25 00:18:29 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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  |                                                                            
  |==================                                                    |  25%
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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 @ Wed Feb 25 00:18:51 2026 -------------------
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d15ed4b3d3/fastq, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/fastq/sample1.fq.gz, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/fastq/sample2.fq.gz, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample1_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample2_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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/RtmpP10EfK/file3a92d15ed4b3d3/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d15ed4b3d3/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 @ Wed Feb 25 00:18:51 2026 ----------
00:18:52 Wed Feb 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d15ed4b3d3/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample1_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample1_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample2_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample2_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample3_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d15ed4b3d3/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d127561f13/config_file_3838673.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 @ Wed Feb 25 00:18:53 2026 ----------------
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/RtmpP10EfK/file3a92d127561f13/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d127561f13/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d127561f13/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d127561f13/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/RtmpP10EfK/file3a92d127561f13/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d127561f13/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/RtmpP10EfK/file3a92d127561f13/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d127561f13/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/RtmpP10EfK/file3a92d127561f13/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d127561f13/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 @ Wed Feb 25 00:18:54 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d127561f13/sampleA_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d127561f13/sampleA_align2genome.bam
/tmp/RtmpP10EfK/file3a92d127561f13/sample1_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d127561f13/sample1_align2genome.bam
/tmp/RtmpP10EfK/file3a92d127561f13/sample2_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d127561f13/sample2_align2genome.bam
/tmp/RtmpP10EfK/file3a92d127561f13/sample3_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d127561f13/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Feb 25 00:19:14 2026 ----------------
00:19:14 Wed Feb 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpP10EfK/file3a92d127561f13/sampleA_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d127561f13/sample1_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d127561f13/sample2_align2genome.bam', and
'/tmp/RtmpP10EfK/file3a92d127561f13/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/RtmpP10EfK/file3a92d127561f13/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.29gene_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, 392445.83Read/s]
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.62gene_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, 1313182.22Read/s]
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.59gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1271617.75Read/s]
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.65gene_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, 755893.89Read/s]
-- Running step: isoform_identification @ Wed Feb 25 00:19:15 2026 -------------
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 @ Wed Feb 25 00:19:38 2026 -------------------
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d127561f13/fastq, /tmp/RtmpP10EfK/file3a92d127561f13/fastq/sample1.fq.gz, /tmp/RtmpP10EfK/file3a92d127561f13/fastq/sample2.fq.gz, /tmp/RtmpP10EfK/file3a92d127561f13/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d127561f13/sampleA_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d127561f13/sample1_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d127561f13/sample2_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d127561f13/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d127561f13/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d127561f13/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d127561f13/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d127561f13/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d127561f13/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d127561f13/sampleA_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d127561f13/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d127561f13/sample1_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d127561f13/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d127561f13/sample2_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d127561f13/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d127561f13/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:19:58 2026 ----------
00:19:58 Wed Feb 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sampleA_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d127561f13/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample1_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d127561f13/sample1_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample2_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d127561f13/sample2_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample3_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d127561f13/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d127561f13/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d11cb56ada/config_file_3838673.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 @ Wed Feb 25 00:20:00 2026 ----------------
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/RtmpP10EfK/file3a92d11cb56ada/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d11cb56ada/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d11cb56ada/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d11cb56ada/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/RtmpP10EfK/file3a92d11cb56ada/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d11cb56ada/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/RtmpP10EfK/file3a92d11cb56ada/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d11cb56ada/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/RtmpP10EfK/file3a92d11cb56ada/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d11cb56ada/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 @ Wed Feb 25 00:20:01 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpP10EfK/file3a92d11cb56ada/sampleA_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d11cb56ada/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpP10EfK/file3a92d11cb56ada/sample3_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d11cb56ada/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Feb 25 00:20:02 2026 ----------------
00:20:02 Wed Feb 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpP10EfK/file3a92d11cb56ada/sampleA_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_align2genome.bam', and
'/tmp/RtmpP10EfK/file3a92d11cb56ada/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/RtmpP10EfK/file3a92d11cb56ada/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.67gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 389602.44Read/s]
parsing /tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.32gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1443325.53Read/s]
parsing /tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.64gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1293420.50Read/s]
parsing /tmp/RtmpP10EfK/file3a92d11cb56ada/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.37gene_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, 790244.93Read/s]
-- Running step: isoform_identification @ Wed Feb 25 00:20:03 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:20:03 2026 -------------------
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d11cb56ada/fastq, /tmp/RtmpP10EfK/file3a92d11cb56ada/fastq/sample1.fq.gz, /tmp/RtmpP10EfK/file3a92d11cb56ada/fastq/sample2.fq.gz, /tmp/RtmpP10EfK/file3a92d11cb56ada/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d11cb56ada/sampleA_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d11cb56ada/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d11cb56ada/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d11cb56ada/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpP10EfK/file3a92d11cb56ada/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d11cb56ada/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpP10EfK/file3a92d11cb56ada/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d11cb56ada/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Feb 25 00:20:05 2026 ----------
2026-02-25T05:20:05.882760Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:20:05.883379Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d11cb56ada/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-02-25T05:20:05.883401Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:20:05.883410Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:20:05.883497Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:20:05.883511Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-25T05:20:05.895888Z  INFO oarfish::single_cell: Processed 100 cells.
2026-02-25T05:20:06.416876Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:20:06.417509Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d11cb56ada/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-02-25T05:20:06.417533Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:20:06.417542Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:20:06.417621Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:20:06.417636Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-25T05:20:06.977021Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:20:06.977567Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d11cb56ada/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-02-25T05:20:06.977590Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:20:06.977599Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:20:06.977678Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:20:06.977693Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-25T05:20:07.654509Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:20:07.654928Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d11cb56ada/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-02-25T05:20:07.654949Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:20:07.654957Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:20:07.655037Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:20:07.655051Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d16b7ddd49/config_file_3838673.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 @ Wed Feb 25 00:20:08 2026 ----------------
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/RtmpP10EfK/file3a92d16b7ddd49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d16b7ddd49/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d16b7ddd49/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d16b7ddd49/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/RtmpP10EfK/file3a92d16b7ddd49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d16b7ddd49/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/RtmpP10EfK/file3a92d16b7ddd49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d16b7ddd49/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/RtmpP10EfK/file3a92d16b7ddd49/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d16b7ddd49/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 @ Wed Feb 25 00:20:09 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_align2genome.bam
/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_align2genome.bam
/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_align2genome.bam
/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Feb 25 00:20:29 2026 ----------------
00:20:29 Wed Feb 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_align2genome.bam', and
'/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_align2genome.bam'
parsing /tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.07gene_group/s]
/home/biocbuild/bbs-3.23-bioc/R/site-library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 398289.21Read/s]
parsing /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 42.60gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1249048.24Read/s]
parsing /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.53gene_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, 1269155.17Read/s]
parsing /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.00gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 730307.84Read/s]
-- Running step: isoform_identification @ Wed Feb 25 00:20:30 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:20:30 2026 -------------------
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d16b7ddd49/fastq, /tmp/RtmpP10EfK/file3a92d16b7ddd49/fastq/sample1.fq.gz, /tmp/RtmpP10EfK/file3a92d16b7ddd49/fastq/sample2.fq.gz, /tmp/RtmpP10EfK/file3a92d16b7ddd49/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:20:51 2026 ----------
2026-02-25T05:20:51.544135Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:20:51.544576Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d16b7ddd49/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-02-25T05:20:51.544599Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:20:51.544608Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:20:51.544694Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:20:51.544708Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-25T05:20:51.556595Z  INFO oarfish::single_cell: Processed 100 cells.
2026-02-25T05:20:52.178060Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:20:52.178552Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-02-25T05:20:52.178576Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:20:52.178589Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:20:52.178671Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:20:52.178686Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-25T05:20:52.740586Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:20:52.741063Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-02-25T05:20:52.741084Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:20:52.741093Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:20:52.741172Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:20:52.741186Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-02-25T05:20:53.269300Z  INFO oarfish: setting user-provided filter parameters.
2026-02-25T05:20:53.269840Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpP10EfK/file3a92d16b7ddd49/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-02-25T05:20:53.269864Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-02-25T05:20:53.269872Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-02-25T05:20:53.269950Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-02-25T05:20:53.269964Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d134e740b9/config_file_3838673.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 @ Wed Feb 25 00:20:54 2026 ----------------
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/RtmpP10EfK/file3a92d134e740b9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d134e740b9/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d134e740b9/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d134e740b9/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 @ Wed Feb 25 00:20:57 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/sample1_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/sample2_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/sample3_matched_reads.fastq.gz -> /tmp/RtmpP10EfK/file3a92d134e740b9/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 @ Wed Feb 25 00:20:59 2026 ----------------
00:20:59 Wed Feb 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d134e740b9/sample1_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d134e740b9/sample2_align2genome.bam', and
'/tmp/RtmpP10EfK/file3a92d134e740b9/sample3_align2genome.bam'
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.20gene_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, 415953.03Read/s]
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 39.34gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1253827.57Read/s]
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.26gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1281877.75Read/s]
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 25.58gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 691125.76Read/s]
-- Running step: isoform_identification @ Wed Feb 25 00:21:00 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:21:00 2026 -------------------
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d134e740b9/fastq, /tmp/RtmpP10EfK/file3a92d134e740b9/fastq/sample1.fq.gz, /tmp/RtmpP10EfK/file3a92d134e740b9/fastq/sample2.fq.gz, /tmp/RtmpP10EfK/file3a92d134e740b9/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d134e740b9/sample1_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d134e740b9/sample2_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d134e740b9/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d134e740b9/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d134e740b9/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d134e740b9/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d134e740b9/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/RtmpP10EfK/file3a92d134e740b9/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpP10EfK/file3a92d134e740b9/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 @ Wed Feb 25 00:21:01 2026 ----------
00:21:01 Wed Feb 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d134e740b9/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample1_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d134e740b9/sample1_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample2_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d134e740b9/sample2_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample3_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d134e740b9/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d134e740b9/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpP10EfK/file3a92d12bef3356/config_file_3838673.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 @ Wed Feb 25 00:21:04 2026 ----------------
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/RtmpP10EfK/file3a92d12bef3356/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d12bef3356/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d12bef3356/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpP10EfK/file3a92d12bef3356/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/RtmpP10EfK/file3a92d12bef3356/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d12bef3356/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/RtmpP10EfK/file3a92d12bef3356/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d12bef3356/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/RtmpP10EfK/file3a92d12bef3356/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpP10EfK/file3a92d12bef3356/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 @ Wed Feb 25 00:21:05 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_align2genome.bam
/tmp/RtmpP10EfK/file3a92d12bef3356/sample1_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d12bef3356/sample1_align2genome.bam
/tmp/RtmpP10EfK/file3a92d12bef3356/sample2_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d12bef3356/sample2_align2genome.bam
/tmp/RtmpP10EfK/file3a92d12bef3356/sample3_matched_reads.fastq.gz ->/tmp/RtmpP10EfK/file3a92d12bef3356/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Feb 25 00:21:25 2026 ----------------
00:21:25 Wed Feb 25 2026 quantify genes 
Using BAM(s): '/tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d12bef3356/sample1_align2genome.bam',
'/tmp/RtmpP10EfK/file3a92d12bef3356/sample2_align2genome.bam', and
'/tmp/RtmpP10EfK/file3a92d12bef3356/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/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, 432348.99Read/s]
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1389394.46Read/s]
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.60gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1288810.23Read/s]
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.43gene_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, 749678.99Read/s]
-- Running step: isoform_identification @ Wed Feb 25 00:21:26 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Feb 25 00:21:27 2026 -------------------
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d12bef3356/fastq, /tmp/RtmpP10EfK/file3a92d12bef3356/fastq/sample1.fq.gz, /tmp/RtmpP10EfK/file3a92d12bef3356/fastq/sample2.fq.gz, /tmp/RtmpP10EfK/file3a92d12bef3356/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d12bef3356/sample1_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d12bef3356/sample2_matched_reads.fastq.gz, /tmp/RtmpP10EfK/file3a92d12bef3356/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d12bef3356/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d12bef3356/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpP10EfK/file3a92d12bef3356/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d12bef3356/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d12bef3356/sample1_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d12bef3356/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d12bef3356/sample2_realign2transcript.bam
/tmp/RtmpP10EfK/file3a92d12bef3356/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpP10EfK/file3a92d12bef3356/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Feb 25 00:21:46 2026 ----------
00:21:46 Wed Feb 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d12bef3356/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample1_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d12bef3356/sample1_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample2_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d12bef3356/sample2_realign2transcript.bamdone
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample3_realign2transcript.bam...
parsing /tmp/RtmpP10EfK/file3a92d12bef3356/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpP10EfK/file3a92d12bef3356/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 182 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 182 | 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 
752.280  46.298 788.980