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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.4.2.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.2 (2025-10-31)
* 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 24.04.4 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.4.2’
* 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-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘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-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     26.624  0.769  27.394
blaze                         4.856 16.619  12.876
find_variants                19.345  0.067  18.799
bulk_long_pipeline            2.362 13.522   2.492
sc_long_multisample_pipeline  8.319  6.428   8.558
sc_plot_genotype             10.843  1.025  10.709
MultiSampleSCPipeline        10.233  0.625  11.212
sc_DTU_analysis               7.255  2.230   7.331
plot_isoform_heatmap          7.030  0.102   7.131
create_sce_from_dir           3.500  2.658   3.667
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

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

##############################################################################
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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
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* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.4.2’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

R version 4.5.2 (2025-10-31) -- "[Not] Part in a Rumble"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file394816560e6393/config_file_3754006.json 
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file394816560e6393/config_file_3754006.json 
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file394816560e6393/config_file_3754006.json 
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481611b0b358/config_file_3754006.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/RtmpmMr1XJ/file3948161c7b9e6f/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpmMr1XJ/file39481659c31e60/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpmMr1XJ/file39481659c31e60/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file39481622b52003/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file39481622b52003/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file39481622b52003/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file39481622b52003/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/RtmpmMr1XJ/file394816df40874/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file3948163db4bf52/config_file_3754006.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Apr  7 23:40:17 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpmMr1XJ/file3948163db4bf52/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpmMr1XJ/file3948163db4bf52/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpmMr1XJ/file3948163db4bf52/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Apr  7 23:40:19 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |===============================================                       |  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 @ Tue Apr  7 23:40:43 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpmMr1XJ/file3948163db4bf52/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpmMr1XJ/file3948163db4bf52/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpmMr1XJ/file3948163db4bf52/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Tue Apr  7 23:40:43 2026 ----------
2026-04-08T03:40:43.851056Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:40:43.851421Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948163db4bf52/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:40:43.851432Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:40:43.851445Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:40:43.851504Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:40:43.851509Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:40:43.853057Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:40:43.853174Z  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    │
╰─────────────────────────────────┴───────╯

2026-04-08T03:40:43.853194Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-04-08T03:40:43.853196Z  INFO oarfish::bulk: number of aligned reads : 96
2026-04-08T03:40:43.853198Z  INFO oarfish::bulk: number of unique alignments : 86
2026-04-08T03:40:43.853909Z  INFO oarfish: oarfish completed successfully.
2026-04-08T03:40:43.860712Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:40:43.861033Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948163db4bf52/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:40:43.861041Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:40:43.861044Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:40:43.861099Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:40:43.861104Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:40:43.862724Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:40:43.862844Z  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    │
╰─────────────────────────────────┴───────╯

2026-04-08T03:40:43.862868Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-04-08T03:40:43.862870Z  INFO oarfish::bulk: number of aligned reads : 95
2026-04-08T03:40:43.862881Z  INFO oarfish::bulk: number of unique alignments : 82
2026-04-08T03:40:43.863481Z  INFO oarfish: oarfish completed successfully.
2026-04-08T03:40:43.870261Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:40:43.870587Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948163db4bf52/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:40:43.870598Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:40:43.870600Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:40:43.870657Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:40:43.870662Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:40:43.873386Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-08T03:40:43.873547Z  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   │
╰─────────────────────────────────┴───────╯

2026-04-08T03:40:43.873572Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-04-08T03:40:43.873575Z  INFO oarfish::bulk: number of aligned reads : 179
2026-04-08T03:40:43.873577Z  INFO oarfish::bulk: number of unique alignments : 143
2026-04-08T03:40:43.874296Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file3948168a97182/config_file_3754006.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Apr  7 23:40:44 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpmMr1XJ/file3948168a97182/sample1_align2genome.bam
sample2 ->/tmp/RtmpmMr1XJ/file3948168a97182/sample2_align2genome.bam
sample3 ->/tmp/RtmpmMr1XJ/file3948168a97182/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Apr  7 23:41:04 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Tue Apr  7 23:41:25 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpmMr1XJ/file3948168a97182/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpmMr1XJ/file3948168a97182/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpmMr1XJ/file3948168a97182/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:41:45 2026 ----------
2026-04-08T03:41:45.223136Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:41:45.223560Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948168a97182/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:41:45.223573Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:41:45.223577Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:41:45.223649Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:41:45.223655Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:41:45.225305Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:41:45.225444Z  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    │
╰─────────────────────────────────┴───────╯

2026-04-08T03:41:45.225467Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-04-08T03:41:45.225471Z  INFO oarfish::bulk: number of aligned reads : 96
2026-04-08T03:41:45.225474Z  INFO oarfish::bulk: number of unique alignments : 86
2026-04-08T03:41:45.226117Z  INFO oarfish: oarfish completed successfully.
2026-04-08T03:41:45.237413Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:41:45.238117Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948168a97182/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:41:45.238130Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:41:45.238148Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:41:45.238213Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:41:45.238218Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:41:45.240082Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:41:45.240243Z  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    │
╰─────────────────────────────────┴───────╯

2026-04-08T03:41:45.240282Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-04-08T03:41:45.240287Z  INFO oarfish::bulk: number of aligned reads : 95
2026-04-08T03:41:45.240289Z  INFO oarfish::bulk: number of unique alignments : 82
2026-04-08T03:41:45.240920Z  INFO oarfish: oarfish completed successfully.
2026-04-08T03:41:45.252343Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:41:45.252765Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948168a97182/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:41:45.252774Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:41:45.252778Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:41:45.252839Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:41:45.252844Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:41:45.255601Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-04-08T03:41:45.255779Z  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   │
╰─────────────────────────────────┴───────╯

2026-04-08T03:41:45.255809Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-04-08T03:41:45.255812Z  INFO oarfish::bulk: number of aligned reads : 179
2026-04-08T03:41:45.255828Z  INFO oarfish::bulk: number of unique alignments : 143
2026-04-08T03:41:45.256560Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file3948166b436b20/config_file_3754006.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Apr  7 23:41:45 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpmMr1XJ/file3948166b436b20/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpmMr1XJ/file3948166b436b20/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpmMr1XJ/file3948166b436b20/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Apr  7 23:41:46 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 @ Tue Apr  7 23:42:04 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpmMr1XJ/file3948166b436b20/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpmMr1XJ/file3948166b436b20/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpmMr1XJ/file3948166b436b20/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Apr  7 23:42:04 2026 ----------
23:42:04 Tue Apr 07 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/RtmpmMr1XJ/file394816e6dcd73/config_file_3754006.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Apr  7 23:42:05 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpmMr1XJ/file394816e6dcd73/sample1_align2genome.bam
sample2 ->/tmp/RtmpmMr1XJ/file394816e6dcd73/sample2_align2genome.bam
sample3 ->/tmp/RtmpmMr1XJ/file394816e6dcd73/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Apr  7 23:42:26 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Tue Apr  7 23:42:43 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpmMr1XJ/file394816e6dcd73/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpmMr1XJ/file394816e6dcd73/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpmMr1XJ/file394816e6dcd73/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:43:03 2026 ----------
23:43:03 Tue Apr 07 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpmMr1XJ/file3948166b436b20/sample1_realign2transcript.bam', '/tmp/RtmpmMr1XJ/file3948166b436b20/sample2_realign2transcript.bam', '/tmp/RtmpmMr1XJ/file3948166b436b20/sample3_realign2transcript.bam'] /tmp/RtmpmMr1XJ/file3948166b436b20/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/RtmpmMr1XJ/file394816388e4004/config_file_3754006.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Apr  7 23:43:03 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpmMr1XJ/file394816388e4004/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpmMr1XJ/file394816388e4004/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpmMr1XJ/file394816388e4004/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Apr  7 23:43:04 2026 -------------
Inputs:  ['/tmp/RtmpmMr1XJ/file394816e6dcd73/sample1_realign2transcript.bam', '/tmp/RtmpmMr1XJ/file394816e6dcd73/sample2_realign2transcript.bam', '/tmp/RtmpmMr1XJ/file394816e6dcd73/sample3_realign2transcript.bam'] /tmp/RtmpmMr1XJ/file394816e6dcd73/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:43:05 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpmMr1XJ/file394816388e4004/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpmMr1XJ/file394816388e4004/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpmMr1XJ/file394816388e4004/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Tue Apr  7 23:43:05 2026 ----------
2026-04-08T03:43:05.946322Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:43:05.946682Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file394816388e4004/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-04-08T03:43:05.946690Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:43:05.946693Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:43:05.946762Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:43:05.946768Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-08T03:43:05.949234Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:43:05.949363Z  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    │
╰─────────────────────────────────┴───────╯

2026-04-08T03:43:05.949385Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-04-08T03:43:05.949387Z  INFO oarfish::bulk: number of aligned reads : 98
2026-04-08T03:43:05.949389Z  INFO oarfish::bulk: number of unique alignments : 86
2026-04-08T03:43:05.949978Z  INFO oarfish: oarfish completed successfully.
2026-04-08T03:43:05.956808Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:43:05.957144Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file394816388e4004/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-04-08T03:43:05.957152Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:43:05.957155Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:43:05.957223Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:43:05.957229Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-08T03:43:05.959725Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:43:05.959858Z  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    │
╰─────────────────────────────────┴───────╯

2026-04-08T03:43:05.959881Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-04-08T03:43:05.959884Z  INFO oarfish::bulk: number of aligned reads : 97
2026-04-08T03:43:05.959886Z  INFO oarfish::bulk: number of unique alignments : 79
2026-04-08T03:43:05.960495Z  INFO oarfish: oarfish completed successfully.
2026-04-08T03:43:05.967433Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:43:05.967934Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file394816388e4004/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-04-08T03:43:05.967942Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:43:05.967944Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:43:05.968008Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:43:05.968014Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-08T03:43:05.972189Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:43:05.972360Z  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   │
╰─────────────────────────────────┴───────╯

2026-04-08T03:43:05.972394Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-04-08T03:43:05.972397Z  INFO oarfish::bulk: number of aligned reads : 187
2026-04-08T03:43:05.972399Z  INFO oarfish::bulk: number of unique alignments : 140
2026-04-08T03:43:05.973083Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481654d82635/config_file_3754006.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Apr  7 23:43:06 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpmMr1XJ/file39481654d82635/sample1_align2genome.bam
sample2 ->/tmp/RtmpmMr1XJ/file39481654d82635/sample2_align2genome.bam
sample3 ->/tmp/RtmpmMr1XJ/file39481654d82635/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Apr  7 23:43:25 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:43:25 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpmMr1XJ/file39481654d82635/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpmMr1XJ/file39481654d82635/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpmMr1XJ/file39481654d82635/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:43:45 2026 ----------
2026-04-08T03:43:45.144200Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:43:45.144588Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file39481654d82635/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-04-08T03:43:45.144599Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:43:45.144602Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:43:45.144673Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:43:45.144681Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-08T03:43:45.147155Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:43:45.147284Z  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    │
╰─────────────────────────────────┴───────╯

2026-04-08T03:43:45.147305Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-04-08T03:43:45.147309Z  INFO oarfish::bulk: number of aligned reads : 98
2026-04-08T03:43:45.147312Z  INFO oarfish::bulk: number of unique alignments : 86
2026-04-08T03:43:45.147891Z  INFO oarfish: oarfish completed successfully.
2026-04-08T03:43:45.156713Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:43:45.157115Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file39481654d82635/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-04-08T03:43:45.157125Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:43:45.157129Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:43:45.157206Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:43:45.157213Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-08T03:43:45.159899Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:43:45.160020Z  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    │
╰─────────────────────────────────┴───────╯

2026-04-08T03:43:45.160044Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-04-08T03:43:45.160047Z  INFO oarfish::bulk: number of aligned reads : 97
2026-04-08T03:43:45.160049Z  INFO oarfish::bulk: number of unique alignments : 79
2026-04-08T03:43:45.160650Z  INFO oarfish: oarfish completed successfully.
2026-04-08T03:43:45.169080Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:43:45.169440Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file39481654d82635/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-04-08T03:43:45.169450Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:43:45.169453Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:43:45.169520Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:43:45.169526Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-08T03:43:45.173980Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-04-08T03:43:45.174121Z  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   │
╰─────────────────────────────────┴───────╯

2026-04-08T03:43:45.174147Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-04-08T03:43:45.174150Z  INFO oarfish::bulk: number of aligned reads : 187
2026-04-08T03:43:45.174152Z  INFO oarfish::bulk: number of unique alignments : 140
2026-04-08T03:43:45.174865Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481653a39e19/config_file_3754006.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Apr  7 23:43:45 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpmMr1XJ/file39481653a39e19/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpmMr1XJ/file39481653a39e19/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpmMr1XJ/file39481653a39e19/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Apr  7 23:43:46 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:43:46 2026 -------------------
Realigning sample sample1 -> /tmp/RtmpmMr1XJ/file39481653a39e19/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpmMr1XJ/file39481653a39e19/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpmMr1XJ/file39481653a39e19/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Apr  7 23:43:47 2026 ----------
23:43:47 Tue Apr 07 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/RtmpmMr1XJ/file3948162a9cb121/config_file_3754006.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Apr  7 23:43:48 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpmMr1XJ/file3948162a9cb121/sample1_align2genome.bam
sample2 ->/tmp/RtmpmMr1XJ/file3948162a9cb121/sample2_align2genome.bam
sample3 ->/tmp/RtmpmMr1XJ/file3948162a9cb121/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Apr  7 23:44:07 2026 -------------
Inputs:  ['/tmp/RtmpmMr1XJ/file39481653a39e19/sample1_realign2transcript.bam', '/tmp/RtmpmMr1XJ/file39481653a39e19/sample2_realign2transcript.bam', '/tmp/RtmpmMr1XJ/file39481653a39e19/sample3_realign2transcript.bam'] /tmp/RtmpmMr1XJ/file39481653a39e19/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:44:08 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpmMr1XJ/file3948162a9cb121/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpmMr1XJ/file3948162a9cb121/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpmMr1XJ/file3948162a9cb121/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:44:27 2026 ----------
23:44:27 Tue Apr 07 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/RtmpmMr1XJ/file3948162af41961/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:44:28 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/RtmpmMr1XJ/file3948162af41961/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Tue Apr  7 23:44:28 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpmMr1XJ/file3948162af41961/matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948162af41961/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Apr  7 23:44:28 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 @ Tue Apr  7 23:44:38 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948162af41961/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948162af41961/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpmMr1XJ/file3948162af41961/matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948162af41961/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Tue Apr  7 23:44:38 2026 ----------
2026-04-08T03:44:38.727869Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:44:38.728326Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948162af41961/realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:44:38.728339Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:44:38.728342Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:44:38.728405Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:44:38.728410Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:44:38.734803Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481664a824f6/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:44:39 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/RtmpmMr1XJ/file39481664a824f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Tue Apr  7 23:44:39 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpmMr1XJ/file39481664a824f6/matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481664a824f6/align2genome.bam
-- Running step: isoform_identification @ Tue Apr  7 23:44:58 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 @ Tue Apr  7 23:45:07 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481664a824f6/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481664a824f6/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpmMr1XJ/file39481664a824f6/matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481664a824f6/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:45:27 2026 ----------
2026-04-08T03:45:27.205162Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:45:27.205703Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file39481664a824f6/realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:45:27.205714Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:45:27.205718Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:45:27.205777Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:45:27.205784Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:45:27.212320Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481650c37aa/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:45:27 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/RtmpmMr1XJ/file39481650c37aa/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Tue Apr  7 23:45:27 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpmMr1XJ/file39481650c37aa/matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file39481650c37aa/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Apr  7 23:45:28 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 @ Tue Apr  7 23:45:38 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481650c37aa/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481650c37aa/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpmMr1XJ/file39481650c37aa/matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file39481650c37aa/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Tue Apr  7 23:45:38 2026 ----------
23:45:38 Tue Apr 07 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpmMr1XJ/file3948162a9cb121/sample1_realign2transcript.bam', '/tmp/RtmpmMr1XJ/file3948162a9cb121/sample2_realign2transcript.bam', '/tmp/RtmpmMr1XJ/file3948162a9cb121/sample3_realign2transcript.bam'] /tmp/RtmpmMr1XJ/file3948162a9cb121/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/RtmpmMr1XJ/file3948162c15a5c6/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:45:39 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/RtmpmMr1XJ/file3948162c15a5c6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Tue Apr  7 23:45:39 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpmMr1XJ/file3948162c15a5c6/matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162c15a5c6/align2genome.bam
-- Running step: isoform_identification @ Tue Apr  7 23:45:58 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 @ Tue Apr  7 23:46:07 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948162c15a5c6/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948162c15a5c6/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpmMr1XJ/file3948162c15a5c6/matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162c15a5c6/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:46:27 2026 ----------
23:46:27 Tue Apr 07 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/RtmpmMr1XJ/file39481621d17996/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:46:28 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/RtmpmMr1XJ/file39481621d17996/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Tue Apr  7 23:46:28 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpmMr1XJ/file39481621d17996/matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file39481621d17996/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Apr  7 23:46:28 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 @ Tue Apr  7 23:46:29 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481621d17996/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481621d17996/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpmMr1XJ/file39481621d17996/matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file39481621d17996/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Tue Apr  7 23:46:29 2026 ----------
2026-04-08T03:46:29.415712Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:46:29.416124Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file39481621d17996/realign2transcript.bam, contains 10 reference sequences.
2026-04-08T03:46:29.416131Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:46:29.416134Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:46:29.416207Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:46:29.416214Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-08T03:46:29.425821Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481635e6fcb2/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:46:30 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/RtmpmMr1XJ/file39481635e6fcb2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Tue Apr  7 23:46:30 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpmMr1XJ/file39481635e6fcb2/matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481635e6fcb2/align2genome.bam
-- Running step: isoform_identification @ Tue Apr  7 23:46:48 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:46:48 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481635e6fcb2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481635e6fcb2/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpmMr1XJ/file39481635e6fcb2/matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481635e6fcb2/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:47:08 2026 ----------
2026-04-08T03:47:08.214520Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:47:08.214985Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file39481635e6fcb2/realign2transcript.bam, contains 10 reference sequences.
2026-04-08T03:47:08.214997Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:47:08.215001Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:47:08.215078Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:47:08.215086Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-04-08T03:47:08.224952Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481670a71703/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:47:09 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/RtmpmMr1XJ/file39481670a71703/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Tue Apr  7 23:47:09 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpmMr1XJ/file39481670a71703/matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file39481670a71703/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Apr  7 23:47:09 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:47:09 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481670a71703/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481670a71703/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpmMr1XJ/file39481670a71703/matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file39481670a71703/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Tue Apr  7 23:47:10 2026 ----------
23:47:10 Tue Apr 07 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481643ee3584/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:47:11 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/RtmpmMr1XJ/file39481643ee3584/bc_allow.tsv
Number of known barcodes: 143
Processing file: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
Searching for barcodes...
Number of reads processed: 393
Number of reads where at least one barcode was found: 368
Number of reads with exactly one barcode match: 364
Number of chimera reads: 1
All done!
Reads	Barcodes
10	2
9	2
8	5
7	4
6	3
5	7
4	14
3	14
2	29
1	57
-- Running step: genome_alignment @ Tue Apr  7 23:47:11 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpmMr1XJ/file39481643ee3584/matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481643ee3584/align2genome.bam
-- Running step: isoform_identification @ Tue Apr  7 23:47:29 2026 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:47:29 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481643ee3584/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481643ee3584/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpmMr1XJ/file39481643ee3584/matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481643ee3584/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:47:47 2026 ----------
23:47:47 Tue Apr 07 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file394816131600c2/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:47:48 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/RtmpmMr1XJ/file394816131600c2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file394816131600c2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file394816131600c2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file394816131600c2/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/RtmpmMr1XJ/file394816131600c2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file394816131600c2/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/RtmpmMr1XJ/file394816131600c2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file394816131600c2/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/RtmpmMr1XJ/file394816131600c2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file394816131600c2/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Tue Apr  7 23:47:49 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpmMr1XJ/file394816131600c2/sampleA_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file394816131600c2/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpmMr1XJ/file394816131600c2/sample1_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file394816131600c2/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpmMr1XJ/file394816131600c2/sample2_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file394816131600c2/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpmMr1XJ/file394816131600c2/sample3_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file394816131600c2/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Apr  7 23:47:51 2026 ----------------
23:47:51 Tue Apr 07 2026 quantify genes 
Using BAM(s): '/tmp/RtmpmMr1XJ/file394816131600c2/sampleA_align2genome.bam',
'/tmp/RtmpmMr1XJ/file394816131600c2/sample1_align2genome.bam',
'/tmp/RtmpmMr1XJ/file394816131600c2/sample2_align2genome.bam', and
'/tmp/RtmpmMr1XJ/file394816131600c2/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpmMr1XJ/file394816131600c2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 443973.24Read/s]
parsing /tmp/RtmpmMr1XJ/file394816131600c2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1441936.19Read/s]
parsing /tmp/RtmpmMr1XJ/file394816131600c2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1392161.44Read/s]
parsing /tmp/RtmpmMr1XJ/file394816131600c2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 738121.92Read/s]
-- Running step: isoform_identification @ Tue Apr  7 23:47:52 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 @ Tue Apr  7 23:48:14 2026 -------------------
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file394816131600c2/fastq, /tmp/RtmpmMr1XJ/file394816131600c2/fastq/sample1.fq.gz, /tmp/RtmpmMr1XJ/file394816131600c2/fastq/sample2.fq.gz, /tmp/RtmpmMr1XJ/file394816131600c2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file394816131600c2/sampleA_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file394816131600c2/sample1_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file394816131600c2/sample2_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file394816131600c2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file394816131600c2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file394816131600c2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file394816131600c2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file394816131600c2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpmMr1XJ/file394816131600c2/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file394816131600c2/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpmMr1XJ/file394816131600c2/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file394816131600c2/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpmMr1XJ/file394816131600c2/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file394816131600c2/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpmMr1XJ/file394816131600c2/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file394816131600c2/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Tue Apr  7 23:48:15 2026 ----------
2026-04-08T03:48:15.706741Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:48:15.707089Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file394816131600c2/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:48:15.707096Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:48:15.707099Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:48:15.707152Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:48:15.707157Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:48:15.712755Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-08T03:48:15.996162Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:48:15.996529Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file394816131600c2/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:48:15.996541Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:48:15.996544Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:48:15.996605Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:48:15.996611Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:48:16.299706Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:48:16.300079Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file394816131600c2/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:48:16.300086Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:48:16.300089Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:48:16.300143Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:48:16.300148Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:48:16.574412Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:48:16.574865Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file394816131600c2/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:48:16.574873Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:48:16.574876Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:48:16.574928Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:48:16.574934Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file3948165c4c6108/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:48:17 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/RtmpmMr1XJ/file3948165c4c6108/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948165c4c6108/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948165c4c6108/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948165c4c6108/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/RtmpmMr1XJ/file3948165c4c6108/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948165c4c6108/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/RtmpmMr1XJ/file3948165c4c6108/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948165c4c6108/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/RtmpmMr1XJ/file3948165c4c6108/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948165c4c6108/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Tue Apr  7 23:48:17 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_align2genome.bam
/tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_align2genome.bam
/tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_align2genome.bam
/tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Apr  7 23:48:37 2026 ----------------
23:48:37 Tue Apr 07 2026 quantify genes 
Using BAM(s): '/tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_align2genome.bam', and
'/tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_align2genome.bam'
parsing /tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.87gene_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, 403748.80Read/s]
parsing /tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 45.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1391422.51Read/s]
parsing /tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.50gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1403716.20Read/s]
parsing /tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.60gene_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, 805543.52Read/s]
-- Running step: isoform_identification @ Tue Apr  7 23:48:38 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Tue Apr  7 23:49:00 2026 -------------------
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948165c4c6108/fastq, /tmp/RtmpmMr1XJ/file3948165c4c6108/fastq/sample1.fq.gz, /tmp/RtmpmMr1XJ/file3948165c4c6108/fastq/sample2.fq.gz, /tmp/RtmpmMr1XJ/file3948165c4c6108/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:49:19 2026 ----------
2026-04-08T03:49:19.025263Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:49:19.025863Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948165c4c6108/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:49:19.025875Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:49:19.025880Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:49:19.025934Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:49:19.025940Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:49:19.031728Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-08T03:49:19.393924Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:49:19.394637Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948165c4c6108/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:49:19.394650Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:49:19.394655Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:49:19.394713Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:49:19.394720Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:49:19.768671Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:49:19.769175Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948165c4c6108/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:49:19.769182Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:49:19.769185Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:49:19.769246Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:49:19.769251Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-04-08T03:49:20.074063Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:49:20.074671Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948165c4c6108/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-04-08T03:49:20.074683Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:49:20.074686Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:49:20.074742Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:49:20.074748Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file394816457f6650/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:49:20 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/RtmpmMr1XJ/file394816457f6650/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file394816457f6650/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file394816457f6650/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file394816457f6650/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Tue Apr  7 23:49:21 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpmMr1XJ/file394816457f6650/sampleA_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/sample1_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/sample2_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/sample3_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file394816457f6650/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Apr  7 23:49:22 2026 ----------------
23:49:22 Tue Apr 07 2026 quantify genes 
Using BAM(s): '/tmp/RtmpmMr1XJ/file394816457f6650/sampleA_align2genome.bam',
'/tmp/RtmpmMr1XJ/file394816457f6650/sample1_align2genome.bam',
'/tmp/RtmpmMr1XJ/file394816457f6650/sample2_align2genome.bam', and
'/tmp/RtmpmMr1XJ/file394816457f6650/sample3_align2genome.bam'
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 432170.80Read/s]
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.60gene_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, 1486709.20Read/s]
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.00gene_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, 1388658.46Read/s]
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.10gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 789947.27Read/s]
-- Running step: isoform_identification @ Tue Apr  7 23:49:23 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Tue Apr  7 23:49:47 2026 -------------------
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file394816457f6650/fastq, /tmp/RtmpmMr1XJ/file394816457f6650/fastq/sample1.fq.gz, /tmp/RtmpmMr1XJ/file394816457f6650/fastq/sample2.fq.gz, /tmp/RtmpmMr1XJ/file394816457f6650/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file394816457f6650/sampleA_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file394816457f6650/sample1_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file394816457f6650/sample2_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file394816457f6650/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file394816457f6650/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file394816457f6650/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file394816457f6650/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file394816457f6650/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpmMr1XJ/file394816457f6650/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file394816457f6650/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/RtmpmMr1XJ/file394816457f6650/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file394816457f6650/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Apr  7 23:49:48 2026 ----------
23:49:48 Tue Apr 07 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sampleA_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file394816457f6650/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample1_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file394816457f6650/sample1_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample2_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file394816457f6650/sample2_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample3_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file394816457f6650/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file394816457f6650/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file39481651dad684/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:49:49 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/RtmpmMr1XJ/file39481651dad684/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file39481651dad684/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file39481651dad684/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file39481651dad684/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/RtmpmMr1XJ/file39481651dad684/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file39481651dad684/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/RtmpmMr1XJ/file39481651dad684/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file39481651dad684/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/RtmpmMr1XJ/file39481651dad684/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file39481651dad684/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Tue Apr  7 23:49:50 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpmMr1XJ/file39481651dad684/sampleA_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481651dad684/sampleA_align2genome.bam
/tmp/RtmpmMr1XJ/file39481651dad684/sample1_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481651dad684/sample1_align2genome.bam
/tmp/RtmpmMr1XJ/file39481651dad684/sample2_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481651dad684/sample2_align2genome.bam
/tmp/RtmpmMr1XJ/file39481651dad684/sample3_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file39481651dad684/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Apr  7 23:50:09 2026 ----------------
23:50:09 Tue Apr 07 2026 quantify genes 
Using BAM(s): '/tmp/RtmpmMr1XJ/file39481651dad684/sampleA_align2genome.bam',
'/tmp/RtmpmMr1XJ/file39481651dad684/sample1_align2genome.bam',
'/tmp/RtmpmMr1XJ/file39481651dad684/sample2_align2genome.bam', and
'/tmp/RtmpmMr1XJ/file39481651dad684/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/RtmpmMr1XJ/file39481651dad684/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.96gene_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, 458113.50Read/s]
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.53gene_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, 1423148.75Read/s]
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.69gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1378435.65Read/s]
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.45gene_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, 720522.23Read/s]
-- Running step: isoform_identification @ Tue Apr  7 23:50:10 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Tue Apr  7 23:50:32 2026 -------------------
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481651dad684/fastq, /tmp/RtmpmMr1XJ/file39481651dad684/fastq/sample1.fq.gz, /tmp/RtmpmMr1XJ/file39481651dad684/fastq/sample2.fq.gz, /tmp/RtmpmMr1XJ/file39481651dad684/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481651dad684/sampleA_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file39481651dad684/sample1_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file39481651dad684/sample2_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file39481651dad684/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file39481651dad684/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file39481651dad684/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file39481651dad684/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file39481651dad684/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpmMr1XJ/file39481651dad684/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file39481651dad684/sampleA_realign2transcript.bam
/tmp/RtmpmMr1XJ/file39481651dad684/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file39481651dad684/sample1_realign2transcript.bam
/tmp/RtmpmMr1XJ/file39481651dad684/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file39481651dad684/sample2_realign2transcript.bam
/tmp/RtmpmMr1XJ/file39481651dad684/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file39481651dad684/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:50:51 2026 ----------
23:50:51 Tue Apr 07 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sampleA_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file39481651dad684/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample1_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file39481651dad684/sample1_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample2_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file39481651dad684/sample2_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample3_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file39481651dad684/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file39481651dad684/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file3948161a5ac2f0/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:50:53 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/RtmpmMr1XJ/file3948161a5ac2f0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948161a5ac2f0/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948161a5ac2f0/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948161a5ac2f0/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/RtmpmMr1XJ/file3948161a5ac2f0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948161a5ac2f0/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/RtmpmMr1XJ/file3948161a5ac2f0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948161a5ac2f0/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/RtmpmMr1XJ/file3948161a5ac2f0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948161a5ac2f0/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Tue Apr  7 23:50:54 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample3_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Apr  7 23:50:55 2026 ----------------
23:50:55 Tue Apr 07 2026 quantify genes 
Using BAM(s): '/tmp/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_align2genome.bam', and
'/tmp/RtmpmMr1XJ/file3948161a5ac2f0/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/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.60gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 443241.32Read/s]
parsing /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1452723.75Read/s]
parsing /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.33gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1275484.73Read/s]
parsing /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.90gene_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, 778626.27Read/s]
-- Running step: isoform_identification @ Tue Apr  7 23:50:56 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:50:56 2026 -------------------
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948161a5ac2f0/fastq, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/fastq/sample1.fq.gz, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/fastq/sample2.fq.gz, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Tue Apr  7 23:50:58 2026 ----------
2026-04-08T03:50:58.805182Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:50:58.805585Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-04-08T03:50:58.805595Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:50:58.805599Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:50:58.805694Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:50:58.805703Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-04-08T03:50:58.817431Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-08T03:50:59.331918Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:50:59.332380Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-04-08T03:50:59.332391Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:50:59.332394Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:50:59.332472Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:50:59.332479Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-04-08T03:50:59.876405Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:50:59.876762Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-04-08T03:50:59.876773Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:50:59.876777Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:50:59.876867Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:50:59.877020Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-04-08T03:51:00.407242Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:51:00.407715Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948161a5ac2f0/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-04-08T03:51:00.407726Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:51:00.407729Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:51:00.407812Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:51:00.407820Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file3948162abc7308/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:51:01 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/RtmpmMr1XJ/file3948162abc7308/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948162abc7308/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948162abc7308/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948162abc7308/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/RtmpmMr1XJ/file3948162abc7308/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948162abc7308/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/RtmpmMr1XJ/file3948162abc7308/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948162abc7308/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/RtmpmMr1XJ/file3948162abc7308/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948162abc7308/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Tue Apr  7 23:51:01 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_align2genome.bam
/tmp/RtmpmMr1XJ/file3948162abc7308/sample1_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162abc7308/sample1_align2genome.bam
/tmp/RtmpmMr1XJ/file3948162abc7308/sample2_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162abc7308/sample2_align2genome.bam
/tmp/RtmpmMr1XJ/file3948162abc7308/sample3_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162abc7308/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Apr  7 23:51:21 2026 ----------------
23:51:21 Tue Apr 07 2026 quantify genes 
Using BAM(s): '/tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948162abc7308/sample1_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948162abc7308/sample2_align2genome.bam', and
'/tmp/RtmpmMr1XJ/file3948162abc7308/sample3_align2genome.bam'
parsing /tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.99gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 390109.75Read/s]
parsing /tmp/RtmpmMr1XJ/file3948162abc7308/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.26gene_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, 1217292.78Read/s]
parsing /tmp/RtmpmMr1XJ/file3948162abc7308/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, 1193054.96Read/s]
parsing /tmp/RtmpmMr1XJ/file3948162abc7308/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.15gene_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, 744833.07Read/s]
-- Running step: isoform_identification @ Tue Apr  7 23:51:22 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:51:23 2026 -------------------
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948162abc7308/fastq, /tmp/RtmpmMr1XJ/file3948162abc7308/fastq/sample1.fq.gz, /tmp/RtmpmMr1XJ/file3948162abc7308/fastq/sample2.fq.gz, /tmp/RtmpmMr1XJ/file3948162abc7308/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948162abc7308/sample1_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948162abc7308/sample2_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948162abc7308/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948162abc7308/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948162abc7308/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948162abc7308/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948162abc7308/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162abc7308/sample1_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948162abc7308/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162abc7308/sample2_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948162abc7308/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948162abc7308/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:51:43 2026 ----------
2026-04-08T03:51:43.346550Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:51:43.346937Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948162abc7308/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-04-08T03:51:43.346946Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:51:43.346950Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:51:43.347027Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:51:43.347035Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-04-08T03:51:43.358890Z  INFO oarfish::single_cell: Processed 100 cells.
2026-04-08T03:51:44.006923Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:51:44.007320Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948162abc7308/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-04-08T03:51:44.007332Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:51:44.007336Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:51:44.007420Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:51:44.007428Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-04-08T03:51:44.561917Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:51:44.562291Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948162abc7308/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-04-08T03:51:44.562303Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:51:44.562307Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:51:44.562395Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:51:44.562404Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-04-08T03:51:45.075717Z  INFO oarfish: setting user-provided filter parameters.
2026-04-08T03:51:45.076255Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpmMr1XJ/file3948162abc7308/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-04-08T03:51:45.076265Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-04-08T03:51:45.076280Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-04-08T03:51:45.076364Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-04-08T03:51:45.076372Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file3948161faad981/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:51:45 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/RtmpmMr1XJ/file3948161faad981/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948161faad981/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948161faad981/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948161faad981/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Tue Apr  7 23:51:46 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpmMr1XJ/file3948161faad981/sampleA_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/sample1_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/sample2_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/sample3_matched_reads.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161faad981/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Apr  7 23:51:48 2026 ----------------
23:51:48 Tue Apr 07 2026 quantify genes 
Using BAM(s): '/tmp/RtmpmMr1XJ/file3948161faad981/sampleA_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948161faad981/sample1_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948161faad981/sample2_align2genome.bam', and
'/tmp/RtmpmMr1XJ/file3948161faad981/sample3_align2genome.bam'
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.51gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 441449.92Read/s]
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.83gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1447509.66Read/s]
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.04gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1356151.06Read/s]
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.82gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 722906.58Read/s]
-- Running step: isoform_identification @ Tue Apr  7 23:51:49 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:51:49 2026 -------------------
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948161faad981/fastq, /tmp/RtmpmMr1XJ/file3948161faad981/fastq/sample1.fq.gz, /tmp/RtmpmMr1XJ/file3948161faad981/fastq/sample2.fq.gz, /tmp/RtmpmMr1XJ/file3948161faad981/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948161faad981/sampleA_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948161faad981/sample1_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948161faad981/sample2_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948161faad981/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948161faad981/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948161faad981/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948161faad981/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948161faad981/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpmMr1XJ/file3948161faad981/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161faad981/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/RtmpmMr1XJ/file3948161faad981/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpmMr1XJ/file3948161faad981/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Apr  7 23:51:50 2026 ----------
23:51:50 Tue Apr 07 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sampleA_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file3948161faad981/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample1_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file3948161faad981/sample1_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample2_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file3948161faad981/sample2_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample3_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file3948161faad981/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file3948161faad981/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpmMr1XJ/file3948165618e6e2/config_file_3754006.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Apr  7 23:51:53 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/RtmpmMr1XJ/file3948165618e6e2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948165618e6e2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948165618e6e2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpmMr1XJ/file3948165618e6e2/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/RtmpmMr1XJ/file3948165618e6e2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948165618e6e2/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/RtmpmMr1XJ/file3948165618e6e2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948165618e6e2/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/RtmpmMr1XJ/file3948165618e6e2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpmMr1XJ/file3948165618e6e2/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Tue Apr  7 23:51:54 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_align2genome.bam
/tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_align2genome.bam
/tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_align2genome.bam
/tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_matched_reads.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Apr  7 23:52:13 2026 ----------------
23:52:13 Tue Apr 07 2026 quantify genes 
Using BAM(s): '/tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_align2genome.bam',
'/tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_align2genome.bam', and
'/tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 300787.70Read/s]
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.60gene_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, 1239598.06Read/s]
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1076898.43Read/s]
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.09gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 704546.13Read/s]
-- Running step: isoform_identification @ Tue Apr  7 23:52:14 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Tue Apr  7 23:52:14 2026 -------------------
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948165618e6e2/fastq, /tmp/RtmpmMr1XJ/file3948165618e6e2/fastq/sample1.fq.gz, /tmp/RtmpmMr1XJ/file3948165618e6e2/fastq/sample2.fq.gz, /tmp/RtmpmMr1XJ/file3948165618e6e2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_matched_reads.fastq.gz, /tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_realign2transcript.bam
/tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Apr  7 23:52:34 2026 ----------
23:52:34 Tue Apr 07 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file3948165618e6e2/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file3948165618e6e2/sample1_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file3948165618e6e2/sample2_realign2transcript.bamdone
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_realign2transcript.bam...
parsing /tmp/RtmpmMr1XJ/file3948165618e6e2/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpmMr1XJ/file3948165618e6e2/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 
725.687  43.789 758.402