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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.5.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 Patched (2025-08-23 r88802)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.3.5’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     25.054  0.161  25.214
blaze                         4.403 18.087  12.472
find_variants                20.164  0.051  19.612
bulk_long_pipeline            2.336 14.698   2.491
sc_long_multisample_pipeline  7.962  6.769   8.342
MultiSampleSCPipeline        10.179  0.692  11.259
sc_plot_genotype             10.434  0.085   9.367
sc_DTU_analysis               7.555  2.210   7.466
plot_isoform_heatmap          6.991  0.112   7.102
create_sce_from_dir           3.444  2.223   3.600
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

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

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

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

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

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Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c8506705d2333/config_file_1869062.json 
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c8506705d2333/config_file_1869062.json 
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c8506705d2333/config_file_1869062.json 
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85062b44d542/config_file_1869062.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/RtmpNKqhpN/file1c85065dd91108/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/RtmpNKqhpN/file1c8506530c49b2/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/RtmpNKqhpN/file1c8506530c49b2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c850614047858/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c850614047858/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c850614047858/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c850614047858/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/RtmpNKqhpN/file1c8506265e174a/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/RtmpNKqhpN/file1c85066e716fa4/config_file_1869062.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 @ Fri Oct 24 23:53:16 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpNKqhpN/file1c85066e716fa4/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpNKqhpN/file1c85066e716fa4/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpNKqhpN/file1c85066e716fa4/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 23:53:17 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:53:24] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:53:24] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:53:25] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:53:25] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:53:26] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.

[23:53:26] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

    https://xgboost.readthedocs.io/en/latest/tutorials/saving_model.html

  for more details about differences between saving model and serializing.


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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 23:53:41 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpNKqhpN/file1c85066e716fa4/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpNKqhpN/file1c85066e716fa4/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpNKqhpN/file1c85066e716fa4/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 24 23:53:42 2025 ----------
2025-10-25T03:53:42.383743Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:53:42.384304Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85066e716fa4/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T03:53:42.384318Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:53:42.384323Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:53:42.384395Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:53:42.384402Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T03:53:42.385980Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:53:42.386141Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-10-25T03:53:42.386166Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-25T03:53:42.386183Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-25T03:53:42.386186Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-25T03:53:42.386879Z  INFO oarfish: oarfish completed successfully.
2025-10-25T03:53:42.394575Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:53:42.395130Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85066e716fa4/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T03:53:42.395143Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:53:42.395146Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:53:42.395212Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:53:42.395218Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T03:53:42.397263Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:53:42.397396Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-10-25T03:53:42.397427Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-25T03:53:42.397429Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-25T03:53:42.397432Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-25T03:53:42.398048Z  INFO oarfish: oarfish completed successfully.
2025-10-25T03:53:42.405495Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:53:42.405859Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85066e716fa4/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T03:53:42.405867Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:53:42.405871Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:53:42.405932Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:53:42.405937Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T03:53:42.408666Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-25T03:53:42.408843Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-10-25T03:53:42.408884Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-25T03:53:42.408886Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-25T03:53:42.408888Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-25T03:53:42.409618Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c850618db9efc/config_file_1869062.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 @ Fri Oct 24 23:53:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpNKqhpN/file1c850618db9efc/sample1_align2genome.bam
sample2 ->/tmp/RtmpNKqhpN/file1c850618db9efc/sample2_align2genome.bam
sample3 ->/tmp/RtmpNKqhpN/file1c850618db9efc/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 23:54:02 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 23:54:21 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpNKqhpN/file1c850618db9efc/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpNKqhpN/file1c850618db9efc/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpNKqhpN/file1c850618db9efc/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 23:54:40 2025 ----------
2025-10-25T03:54:40.952308Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:54:40.952914Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c850618db9efc/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T03:54:40.952940Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:54:40.952943Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:54:40.953005Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:54:40.953011Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T03:54:40.954592Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:54:40.954764Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-10-25T03:54:40.954788Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-10-25T03:54:40.954791Z  INFO oarfish::bulk: number of aligned reads : 96
2025-10-25T03:54:40.954794Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-25T03:54:40.955502Z  INFO oarfish: oarfish completed successfully.
2025-10-25T03:54:40.964750Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:54:40.965201Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c850618db9efc/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T03:54:40.965213Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:54:40.965216Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:54:40.965287Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:54:40.965292Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T03:54:40.966872Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:54:40.967028Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-10-25T03:54:40.967062Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-10-25T03:54:40.967077Z  INFO oarfish::bulk: number of aligned reads : 95
2025-10-25T03:54:40.967079Z  INFO oarfish::bulk: number of unique alignments : 82
2025-10-25T03:54:40.967734Z  INFO oarfish: oarfish completed successfully.
2025-10-25T03:54:40.976801Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:54:40.977220Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c850618db9efc/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T03:54:40.977234Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:54:40.977237Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:54:40.977310Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:54:40.977316Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T03:54:40.980067Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-10-25T03:54:40.980280Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-10-25T03:54:40.980314Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-10-25T03:54:40.980317Z  INFO oarfish::bulk: number of aligned reads : 179
2025-10-25T03:54:40.980319Z  INFO oarfish::bulk: number of unique alignments : 143
2025-10-25T03:54:40.981073Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c850671834fda/config_file_1869062.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 @ Fri Oct 24 23:54:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpNKqhpN/file1c850671834fda/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpNKqhpN/file1c850671834fda/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpNKqhpN/file1c850671834fda/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 23:54:42 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 23:55:00 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpNKqhpN/file1c850671834fda/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpNKqhpN/file1c850671834fda/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpNKqhpN/file1c850671834fda/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 24 23:55:00 2025 ----------
23:55:00 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85067d810085/config_file_1869062.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 @ Fri Oct 24 23:55:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpNKqhpN/file1c85067d810085/sample1_align2genome.bam
sample2 ->/tmp/RtmpNKqhpN/file1c85067d810085/sample2_align2genome.bam
sample3 ->/tmp/RtmpNKqhpN/file1c85067d810085/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 23:55:20 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 23:55:39 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpNKqhpN/file1c85067d810085/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpNKqhpN/file1c85067d810085/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpNKqhpN/file1c85067d810085/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 23:55:58 2025 ----------
23:55:58 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmpNKqhpN/file1c850671834fda/sample1_realign2transcript.bam', '/tmp/RtmpNKqhpN/file1c850671834fda/sample2_realign2transcript.bam', '/tmp/RtmpNKqhpN/file1c850671834fda/sample3_realign2transcript.bam'] /tmp/RtmpNKqhpN/file1c850671834fda/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/RtmpNKqhpN/file1c85062fda4190/config_file_1869062.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 @ Fri Oct 24 23:55:58 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpNKqhpN/file1c85062fda4190/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpNKqhpN/file1c85062fda4190/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpNKqhpN/file1c85062fda4190/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 23:55:59 2025 -------------
Inputs:  ['/tmp/RtmpNKqhpN/file1c85067d810085/sample1_realign2transcript.bam', '/tmp/RtmpNKqhpN/file1c85067d810085/sample2_realign2transcript.bam', '/tmp/RtmpNKqhpN/file1c85067d810085/sample3_realign2transcript.bam'] /tmp/RtmpNKqhpN/file1c85067d810085/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 @ Fri Oct 24 23:56:00 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpNKqhpN/file1c85062fda4190/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmpNKqhpN/file1c85062fda4190/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmpNKqhpN/file1c85062fda4190/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Fri Oct 24 23:56:00 2025 ----------
2025-10-25T03:56:00.957037Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:56:00.957573Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85062fda4190/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T03:56:00.957585Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:56:00.957589Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:56:00.957668Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:56:00.957676Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T03:56:00.960552Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:56:00.960743Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-10-25T03:56:00.960767Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-25T03:56:00.960770Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-25T03:56:00.960773Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-25T03:56:00.961458Z  INFO oarfish: oarfish completed successfully.
2025-10-25T03:56:00.969865Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:56:00.970268Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85062fda4190/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T03:56:00.970280Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:56:00.970284Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:56:00.970353Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:56:00.970360Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T03:56:00.973061Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:56:00.973219Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-10-25T03:56:00.973254Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-25T03:56:00.973257Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-25T03:56:00.973260Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-25T03:56:00.973925Z  INFO oarfish: oarfish completed successfully.
2025-10-25T03:56:00.981651Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:56:00.982030Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85062fda4190/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T03:56:00.982039Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:56:00.982042Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:56:00.982113Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:56:00.982132Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T03:56:00.986257Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:56:00.986435Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-10-25T03:56:00.986465Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-25T03:56:00.986467Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-25T03:56:00.986469Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-25T03:56:00.987209Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85067efa9325/config_file_1869062.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 @ Fri Oct 24 23:56:01 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpNKqhpN/file1c85067efa9325/sample1_align2genome.bam
sample2 ->/tmp/RtmpNKqhpN/file1c85067efa9325/sample2_align2genome.bam
sample3 ->/tmp/RtmpNKqhpN/file1c85067efa9325/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 23:56:20 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 23:56:20 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpNKqhpN/file1c85067efa9325/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpNKqhpN/file1c85067efa9325/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpNKqhpN/file1c85067efa9325/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 23:56:38 2025 ----------
2025-10-25T03:56:38.913691Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:56:38.914198Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067efa9325/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T03:56:38.914210Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:56:38.914225Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:56:38.914310Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:56:38.914317Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T03:56:38.917131Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:56:38.917276Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-10-25T03:56:38.917299Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-10-25T03:56:38.917302Z  INFO oarfish::bulk: number of aligned reads : 98
2025-10-25T03:56:38.917304Z  INFO oarfish::bulk: number of unique alignments : 86
2025-10-25T03:56:38.917972Z  INFO oarfish: oarfish completed successfully.
2025-10-25T03:56:38.928107Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:56:38.928537Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067efa9325/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T03:56:38.928545Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:56:38.928549Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:56:38.928632Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:56:38.928641Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T03:56:38.931288Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:56:38.931424Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-10-25T03:56:38.931456Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-10-25T03:56:38.931459Z  INFO oarfish::bulk: number of aligned reads : 97
2025-10-25T03:56:38.931470Z  INFO oarfish::bulk: number of unique alignments : 79
2025-10-25T03:56:38.932109Z  INFO oarfish: oarfish completed successfully.
2025-10-25T03:56:38.943312Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:56:38.943710Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067efa9325/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-10-25T03:56:38.943718Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:56:38.943721Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:56:38.943792Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:56:38.943800Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T03:56:38.948298Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-10-25T03:56:38.948485Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-10-25T03:56:38.948519Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-10-25T03:56:38.948522Z  INFO oarfish::bulk: number of aligned reads : 187
2025-10-25T03:56:38.948524Z  INFO oarfish::bulk: number of unique alignments : 140
2025-10-25T03:56:38.949263Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c8506a17c300/config_file_1869062.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 @ Fri Oct 24 23:56:39 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmpNKqhpN/file1c8506a17c300/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmpNKqhpN/file1c8506a17c300/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmpNKqhpN/file1c8506a17c300/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 23:56:40 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 23:56:40 2025 -------------------
Realigning sample sample1 -> /tmp/RtmpNKqhpN/file1c8506a17c300/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmpNKqhpN/file1c8506a17c300/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmpNKqhpN/file1c8506a17c300/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Fri Oct 24 23:56:41 2025 ----------
23:56:41 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85064890d58/config_file_1869062.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 @ Fri Oct 24 23:56:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmpNKqhpN/file1c85064890d58/sample1_align2genome.bam
sample2 ->/tmp/RtmpNKqhpN/file1c85064890d58/sample2_align2genome.bam
sample3 ->/tmp/RtmpNKqhpN/file1c85064890d58/sample3_align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 23:57:01 2025 -------------
Inputs:  ['/tmp/RtmpNKqhpN/file1c8506a17c300/sample1_realign2transcript.bam', '/tmp/RtmpNKqhpN/file1c8506a17c300/sample2_realign2transcript.bam', '/tmp/RtmpNKqhpN/file1c8506a17c300/sample3_realign2transcript.bam'] /tmp/RtmpNKqhpN/file1c8506a17c300/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 @ Fri Oct 24 23:57:01 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmpNKqhpN/file1c85064890d58/sample1_realign2transcript.bam
sample2 ->/tmp/RtmpNKqhpN/file1c85064890d58/sample2_realign2transcript.bam
sample3 ->/tmp/RtmpNKqhpN/file1c85064890d58/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 23:57:19 2025 ----------
23:57:19 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c8506275f67d8/config_file_1869062.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 @ Fri Oct 24 23:57:20 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c8506275f67d8/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 @ Fri Oct 24 23:57:21 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNKqhpN/file1c8506275f67d8/matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c8506275f67d8/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 23:57:21 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 23:57:30 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c8506275f67d8/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c8506275f67d8/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpNKqhpN/file1c8506275f67d8/matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c8506275f67d8/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 @ Fri Oct 24 23:57:30 2025 ----------
2025-10-25T03:57:30.720197Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:57:30.720687Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c8506275f67d8/realign2transcript.bam, contains 5 reference sequences.
2025-10-25T03:57:30.720697Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:57:30.720701Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:57:30.720760Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:57:30.720767Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T03:57:30.727193Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c8506111ff85f/config_file_1869062.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 @ Fri Oct 24 23:57:31 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c8506111ff85f/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 @ Fri Oct 24 23:57:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c8506111ff85f/matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c8506111ff85f/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 23:57:49 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 23:57:58 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c8506111ff85f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c8506111ff85f/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c8506111ff85f/matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c8506111ff85f/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 23:58:17 2025 ----------
2025-10-25T03:58:17.493487Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:58:17.493939Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c8506111ff85f/realign2transcript.bam, contains 5 reference sequences.
2025-10-25T03:58:17.493950Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:58:17.493954Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:58:17.494026Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:58:17.494033Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T03:58:17.501036Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85064354e9ea/config_file_1869062.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 @ Fri Oct 24 23:58:18 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85064354e9ea/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 @ Fri Oct 24 23:58:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNKqhpN/file1c85064354e9ea/matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064354e9ea/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 @ Fri Oct 24 23:58:18 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 23:58:28 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85064354e9ea/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85064354e9ea/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpNKqhpN/file1c85064354e9ea/matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064354e9ea/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 @ Fri Oct 24 23:58:28 2025 ----------
23:58:28 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmpNKqhpN/file1c85064890d58/sample1_realign2transcript.bam', '/tmp/RtmpNKqhpN/file1c85064890d58/sample2_realign2transcript.bam', '/tmp/RtmpNKqhpN/file1c85064890d58/sample3_realign2transcript.bam'] /tmp/RtmpNKqhpN/file1c85064890d58/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/RtmpNKqhpN/file1c850664b3af82/config_file_1869062.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 @ Fri Oct 24 23:58:29 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c850664b3af82/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 @ Fri Oct 24 23:58:29 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c850664b3af82/matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c850664b3af82/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 23:58:46 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Fri Oct 24 23:58:56 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c850664b3af82/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c850664b3af82/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c850664b3af82/matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c850664b3af82/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 23:59:14 2025 ----------
23:59:14 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85065bec6422/config_file_1869062.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 @ Fri Oct 24 23:59:15 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85065bec6422/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 @ Fri Oct 24 23:59:16 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNKqhpN/file1c85065bec6422/matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85065bec6422/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Fri Oct 24 23:59:16 2025 -------------
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 23:59:16 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85065bec6422/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85065bec6422/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpNKqhpN/file1c85065bec6422/matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85065bec6422/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 @ Fri Oct 24 23:59:16 2025 ----------
2025-10-25T03:59:16.959053Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:59:16.959494Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85065bec6422/realign2transcript.bam, contains 10 reference sequences.
2025-10-25T03:59:16.959507Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:59:16.959510Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:59:16.959588Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:59:16.959595Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T03:59:16.969339Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c8506382ae735/config_file_1869062.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 @ Fri Oct 24 23:59:17 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c8506382ae735/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 @ Fri Oct 24 23:59:17 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c8506382ae735/matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c8506382ae735/align2genome.bam
-- Running step: isoform_identification @ Fri Oct 24 23:59:35 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 23:59:36 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c8506382ae735/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c8506382ae735/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c8506382ae735/matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c8506382ae735/realign2transcript.bam
-- Running step: transcript_quantification @ Fri Oct 24 23:59:53 2025 ----------
2025-10-25T03:59:53.386394Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T03:59:53.386851Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c8506382ae735/realign2transcript.bam, contains 10 reference sequences.
2025-10-25T03:59:53.386862Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T03:59:53.386866Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T03:59:53.386952Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T03:59:53.386960Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-10-25T03:59:53.396805Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85065e243f27/config_file_1869062.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 @ Fri Oct 24 23:59:54 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85065e243f27/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 @ Fri Oct 24 23:59:54 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNKqhpN/file1c85065e243f27/matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85065e243f27/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 @ Fri Oct 24 23:59:54 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Fri Oct 24 23:59:55 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85065e243f27/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85065e243f27/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmpNKqhpN/file1c85065e243f27/matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85065e243f27/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 @ Fri Oct 24 23:59:55 2025 ----------
23:59:55 Fri Oct 24 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c8506589d91ad/config_file_1869062.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 @ Fri Oct 24 23:59:56 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c8506589d91ad/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 @ Fri Oct 24 23:59:56 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c8506589d91ad/matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c8506589d91ad/align2genome.bam
-- Running step: isoform_identification @ Sat Oct 25 00:00:13 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 25 00:00:14 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c8506589d91ad/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c8506589d91ad/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c8506589d91ad/matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c8506589d91ad/realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 25 00:00:31 2025 ----------
00:00:31 Sat Oct 25 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85064af7be9b/config_file_1869062.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 25 00:00:32 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85064af7be9b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85064af7be9b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85064af7be9b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85064af7be9b/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/RtmpNKqhpN/file1c85064af7be9b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85064af7be9b/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/RtmpNKqhpN/file1c85064af7be9b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85064af7be9b/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/RtmpNKqhpN/file1c85064af7be9b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85064af7be9b/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sat Oct 25 00:00:33 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 25 00:00:35 2025 ----------------
00:00:35 Sat Oct 25 2025 quantify genes 
Using BAM(s): '/tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_align2genome.bam', and
'/tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.81gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 367753.66Read/s]
parsing /tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.80gene_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, 1110897.34Read/s]
parsing /tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.50gene_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, 1051309.40Read/s]
parsing /tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.90gene_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, 659689.21Read/s]
-- Running step: isoform_identification @ Sat Oct 25 00:00:37 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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  |                                                                            
  |==================                                                    |  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 @ Sat Oct 25 00:01:01 2025 -------------------
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85064af7be9b/fastq, /tmp/RtmpNKqhpN/file1c85064af7be9b/fastq/sample1.fq.gz, /tmp/RtmpNKqhpN/file1c85064af7be9b/fastq/sample2.fq.gz, /tmp/RtmpNKqhpN/file1c85064af7be9b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Oct 25 00:01:02 2025 ----------
2025-10-25T04:01:02.754145Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:01:02.754541Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85064af7be9b/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T04:01:02.754565Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:01:02.754567Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:01:02.754630Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:01:02.754636Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T04:01:02.760261Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-25T04:01:03.054836Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:01:03.055230Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85064af7be9b/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T04:01:03.055241Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:01:03.055245Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:01:03.055317Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:01:03.055323Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T04:01:03.344480Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:01:03.344880Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85064af7be9b/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T04:01:03.344889Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:01:03.344892Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:01:03.344950Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:01:03.344956Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T04:01:03.639910Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:01:03.640509Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85064af7be9b/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T04:01:03.640522Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:01:03.640526Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:01:03.640597Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:01:03.640603Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85067ef82a2c/config_file_1869062.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 25 00:01:04 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85067ef82a2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85067ef82a2c/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85067ef82a2c/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85067ef82a2c/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/RtmpNKqhpN/file1c85067ef82a2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85067ef82a2c/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/RtmpNKqhpN/file1c85067ef82a2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85067ef82a2c/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/RtmpNKqhpN/file1c85067ef82a2c/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85067ef82a2c/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sat Oct 25 00:01:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_align2genome.bam
/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_align2genome.bam
/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_align2genome.bam
/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 25 00:01:25 2025 ----------------
00:01:25 Sat Oct 25 2025 quantify genes 
Using BAM(s): '/tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_align2genome.bam', and
'/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_align2genome.bam'
parsing /tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 18.01gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 343008.18Read/s]
parsing /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.06gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1116694.36Read/s]
parsing /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 44.22gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 978788.39Read/s]
parsing /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 31.65gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 626651.53Read/s]
-- Running step: isoform_identification @ Sat Oct 25 00:01:26 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Oct 25 00:01:50 2025 -------------------
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85067ef82a2c/fastq, /tmp/RtmpNKqhpN/file1c85067ef82a2c/fastq/sample1.fq.gz, /tmp/RtmpNKqhpN/file1c85067ef82a2c/fastq/sample2.fq.gz, /tmp/RtmpNKqhpN/file1c85067ef82a2c/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 25 00:02:08 2025 ----------
2025-10-25T04:02:08.322369Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:02:08.322874Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067ef82a2c/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T04:02:08.322884Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:02:08.322888Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:02:08.322946Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:02:08.322953Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T04:02:08.328800Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-25T04:02:08.696949Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:02:08.697487Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T04:02:08.697498Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:02:08.697503Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:02:08.697564Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:02:08.697571Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T04:02:09.028768Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:02:09.029271Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T04:02:09.029283Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:02:09.029287Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:02:09.029350Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:02:09.029357Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-10-25T04:02:09.385451Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:02:09.385952Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067ef82a2c/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-10-25T04:02:09.385963Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:02:09.385967Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:02:09.386026Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:02:09.386044Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85062a649dea/config_file_1869062.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 25 00:02:09 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85062a649dea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85062a649dea/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85062a649dea/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85062a649dea/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sat Oct 25 00:02:10 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/sample1_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/sample2_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/sample3_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85062a649dea/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 25 00:02:12 2025 ----------------
00:02:12 Sat Oct 25 2025 quantify genes 
Using BAM(s): '/tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85062a649dea/sample1_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85062a649dea/sample2_align2genome.bam', and
'/tmp/RtmpNKqhpN/file1c85062a649dea/sample3_align2genome.bam'
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.29gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 399975.59Read/s]
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1249941.59Read/s]
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.47gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1270693.17Read/s]
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.94gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 666820.99Read/s]
-- Running step: isoform_identification @ Sat Oct 25 00:02:13 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Oct 25 00:02:38 2025 -------------------
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85062a649dea/fastq, /tmp/RtmpNKqhpN/file1c85062a649dea/fastq/sample1.fq.gz, /tmp/RtmpNKqhpN/file1c85062a649dea/fastq/sample2.fq.gz, /tmp/RtmpNKqhpN/file1c85062a649dea/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85062a649dea/sample1_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85062a649dea/sample2_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85062a649dea/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85062a649dea/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85062a649dea/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85062a649dea/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85062a649dea/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/RtmpNKqhpN/file1c85062a649dea/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85062a649dea/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 25 00:02:39 2025 ----------
00:02:39 Sat Oct 25 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85062a649dea/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample1_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85062a649dea/sample1_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample2_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85062a649dea/sample2_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample3_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85062a649dea/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85062a649dea/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85066955820a/config_file_1869062.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 25 00:02:41 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85066955820a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85066955820a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85066955820a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85066955820a/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/RtmpNKqhpN/file1c85066955820a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85066955820a/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/RtmpNKqhpN/file1c85066955820a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85066955820a/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/RtmpNKqhpN/file1c85066955820a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85066955820a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sat Oct 25 00:02:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c85066955820a/sampleA_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85066955820a/sampleA_align2genome.bam
/tmp/RtmpNKqhpN/file1c85066955820a/sample1_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85066955820a/sample1_align2genome.bam
/tmp/RtmpNKqhpN/file1c85066955820a/sample2_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85066955820a/sample2_align2genome.bam
/tmp/RtmpNKqhpN/file1c85066955820a/sample3_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85066955820a/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 25 00:03:02 2025 ----------------
00:03:02 Sat Oct 25 2025 quantify genes 
Using BAM(s): '/tmp/RtmpNKqhpN/file1c85066955820a/sampleA_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85066955820a/sample1_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85066955820a/sample2_align2genome.bam', and
'/tmp/RtmpNKqhpN/file1c85066955820a/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/RtmpNKqhpN/file1c85066955820a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.88gene_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, 379712.48Read/s]
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.37gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1212647.16Read/s]
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.53gene_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, 1015078.41Read/s]
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.95gene_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, 610489.05Read/s]
-- Running step: isoform_identification @ Sat Oct 25 00:03:03 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Sat Oct 25 00:03:27 2025 -------------------
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85066955820a/fastq, /tmp/RtmpNKqhpN/file1c85066955820a/fastq/sample1.fq.gz, /tmp/RtmpNKqhpN/file1c85066955820a/fastq/sample2.fq.gz, /tmp/RtmpNKqhpN/file1c85066955820a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85066955820a/sampleA_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85066955820a/sample1_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85066955820a/sample2_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85066955820a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85066955820a/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85066955820a/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85066955820a/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85066955820a/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c85066955820a/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85066955820a/sampleA_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85066955820a/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85066955820a/sample1_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85066955820a/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85066955820a/sample2_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85066955820a/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85066955820a/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 25 00:03:46 2025 ----------
00:03:46 Sat Oct 25 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sampleA_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85066955820a/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample1_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85066955820a/sample1_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample2_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85066955820a/sample2_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample3_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85066955820a/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85066955820a/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85067c8ceafd/config_file_1869062.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 25 00:03:48 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85067c8ceafd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85067c8ceafd/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85067c8ceafd/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85067c8ceafd/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/RtmpNKqhpN/file1c85067c8ceafd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85067c8ceafd/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/RtmpNKqhpN/file1c85067c8ceafd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85067c8ceafd/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/RtmpNKqhpN/file1c85067c8ceafd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85067c8ceafd/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sat Oct 25 00:03:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNKqhpN/file1c85067c8ceafd/sampleA_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85067c8ceafd/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample3_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 25 00:03:50 2025 ----------------
00:03:50 Sat Oct 25 2025 quantify genes 
Using BAM(s): '/tmp/RtmpNKqhpN/file1c85067c8ceafd/sampleA_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_align2genome.bam', and
'/tmp/RtmpNKqhpN/file1c85067c8ceafd/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/RtmpNKqhpN/file1c85067c8ceafd/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.21gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 374411.20Read/s]
parsing /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.71gene_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, 1332032.52Read/s]
parsing /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.06gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1238134.37Read/s]
parsing /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 723305.51Read/s]
-- Running step: isoform_identification @ Sat Oct 25 00:03:51 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 25 00:03:51 2025 -------------------
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85067c8ceafd/fastq, /tmp/RtmpNKqhpN/file1c85067c8ceafd/fastq/sample1.fq.gz, /tmp/RtmpNKqhpN/file1c85067c8ceafd/fastq/sample2.fq.gz, /tmp/RtmpNKqhpN/file1c85067c8ceafd/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85067c8ceafd/sampleA_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85067c8ceafd/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpNKqhpN/file1c85067c8ceafd/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85067c8ceafd/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Sat Oct 25 00:03:53 2025 ----------
2025-10-25T04:03:53.899758Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:03:53.900238Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067c8ceafd/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T04:03:53.900250Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:03:53.900253Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:03:53.900344Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:03:53.900353Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T04:03:53.912046Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-25T04:03:54.425136Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:03:54.425527Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T04:03:54.425535Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:03:54.425538Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:03:54.425623Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:03:54.425631Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T04:03:54.982138Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:03:54.982690Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T04:03:54.982698Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:03:54.982701Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:03:54.982785Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:03:54.982793Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T04:03:55.562555Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:03:55.563135Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85067c8ceafd/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T04:03:55.563149Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:03:55.563152Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:03:55.563240Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:03:55.563249Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85063d153d16/config_file_1869062.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 25 00:03:56 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85063d153d16/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85063d153d16/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85063d153d16/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85063d153d16/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/RtmpNKqhpN/file1c85063d153d16/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85063d153d16/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/RtmpNKqhpN/file1c85063d153d16/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85063d153d16/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/RtmpNKqhpN/file1c85063d153d16/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85063d153d16/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sat Oct 25 00:03:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_align2genome.bam
/tmp/RtmpNKqhpN/file1c85063d153d16/sample1_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85063d153d16/sample1_align2genome.bam
/tmp/RtmpNKqhpN/file1c85063d153d16/sample2_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85063d153d16/sample2_align2genome.bam
/tmp/RtmpNKqhpN/file1c85063d153d16/sample3_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85063d153d16/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 25 00:04:16 2025 ----------------
00:04:16 Sat Oct 25 2025 quantify genes 
Using BAM(s): '/tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85063d153d16/sample1_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85063d153d16/sample2_align2genome.bam', and
'/tmp/RtmpNKqhpN/file1c85063d153d16/sample3_align2genome.bam'
parsing /tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.12gene_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, 334548.70Read/s]
parsing /tmp/RtmpNKqhpN/file1c85063d153d16/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 41.57gene_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, 1022901.18Read/s]
parsing /tmp/RtmpNKqhpN/file1c85063d153d16/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 47.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1065409.47Read/s]
parsing /tmp/RtmpNKqhpN/file1c85063d153d16/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.56gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 612521.76Read/s]
-- Running step: isoform_identification @ Sat Oct 25 00:04:17 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 25 00:04:17 2025 -------------------
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85063d153d16/fastq, /tmp/RtmpNKqhpN/file1c85063d153d16/fastq/sample1.fq.gz, /tmp/RtmpNKqhpN/file1c85063d153d16/fastq/sample2.fq.gz, /tmp/RtmpNKqhpN/file1c85063d153d16/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85063d153d16/sample1_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85063d153d16/sample2_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85063d153d16/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85063d153d16/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85063d153d16/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85063d153d16/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85063d153d16/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85063d153d16/sample1_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85063d153d16/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85063d153d16/sample2_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85063d153d16/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85063d153d16/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 25 00:04:36 2025 ----------
2025-10-25T04:04:36.392171Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:04:36.392612Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85063d153d16/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T04:04:36.392621Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:04:36.392623Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:04:36.392711Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:04:36.392730Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T04:04:36.405101Z  INFO oarfish::single_cell: Processed 100 cells.
2025-10-25T04:04:37.094358Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:04:37.094762Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85063d153d16/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T04:04:37.094771Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:04:37.094775Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:04:37.094865Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:04:37.094874Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T04:04:37.806075Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:04:37.806587Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85063d153d16/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T04:04:37.806600Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:04:37.806603Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:04:37.806692Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:04:37.806700Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-10-25T04:04:38.410095Z  INFO oarfish: setting user-provided filter parameters.
2025-10-25T04:04:38.410613Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmpNKqhpN/file1c85063d153d16/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-10-25T04:04:38.410626Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-10-25T04:04:38.410629Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-10-25T04:04:38.410719Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-10-25T04:04:38.410727Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c850617db10a3/config_file_1869062.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 25 00:04:39 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c850617db10a3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c850617db10a3/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c850617db10a3/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c850617db10a3/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sat Oct 25 00:04:40 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/sample1_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/sample2_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/sample3_matched_reads.fastq.gz -> /tmp/RtmpNKqhpN/file1c850617db10a3/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Sat Oct 25 00:04:41 2025 ----------------
00:04:41 Sat Oct 25 2025 quantify genes 
Using BAM(s): '/tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c850617db10a3/sample1_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c850617db10a3/sample2_align2genome.bam', and
'/tmp/RtmpNKqhpN/file1c850617db10a3/sample3_align2genome.bam'
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.69gene_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, 362565.61Read/s]
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.42gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  6.41gene_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, 1206646.72Read/s]
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.05gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 826235.92Read/s]
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.44gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 607729.22Read/s]
-- Running step: isoform_identification @ Sat Oct 25 00:04:42 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 25 00:04:43 2025 -------------------
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c850617db10a3/fastq, /tmp/RtmpNKqhpN/file1c850617db10a3/fastq/sample1.fq.gz, /tmp/RtmpNKqhpN/file1c850617db10a3/fastq/sample2.fq.gz, /tmp/RtmpNKqhpN/file1c850617db10a3/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c850617db10a3/sample1_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c850617db10a3/sample2_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c850617db10a3/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c850617db10a3/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c850617db10a3/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c850617db10a3/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c850617db10a3/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/RtmpNKqhpN/file1c850617db10a3/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmpNKqhpN/file1c850617db10a3/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Sat Oct 25 00:04:44 2025 ----------
00:04:44 Sat Oct 25 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c850617db10a3/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample1_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c850617db10a3/sample1_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample2_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c850617db10a3/sample2_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample3_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c850617db10a3/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c850617db10a3/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmpNKqhpN/file1c85069896c3d/config_file_1869062.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Sat Oct 25 00:04:47 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmpNKqhpN/file1c85069896c3d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85069896c3d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85069896c3d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmpNKqhpN/file1c85069896c3d/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/RtmpNKqhpN/file1c85069896c3d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85069896c3d/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/RtmpNKqhpN/file1c85069896c3d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85069896c3d/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/RtmpNKqhpN/file1c85069896c3d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmpNKqhpN/file1c85069896c3d/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Sat Oct 25 00:04:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_align2genome.bam
/tmp/RtmpNKqhpN/file1c85069896c3d/sample1_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85069896c3d/sample1_align2genome.bam
/tmp/RtmpNKqhpN/file1c85069896c3d/sample2_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85069896c3d/sample2_align2genome.bam
/tmp/RtmpNKqhpN/file1c85069896c3d/sample3_matched_reads.fastq.gz ->/tmp/RtmpNKqhpN/file1c85069896c3d/sample3_align2genome.bam
-- Running step: gene_quantification @ Sat Oct 25 00:05:07 2025 ----------------
00:05:07 Sat Oct 25 2025 quantify genes 
Using BAM(s): '/tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85069896c3d/sample1_align2genome.bam',
'/tmp/RtmpNKqhpN/file1c85069896c3d/sample2_align2genome.bam', and
'/tmp/RtmpNKqhpN/file1c85069896c3d/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.90gene_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, 298790.68Read/s]
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.13gene_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, 1105276.69Read/s]
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.30gene_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, 1191563.64Read/s]
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 24.90gene_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, 638596.83Read/s]
-- Running step: isoform_identification @ Sat Oct 25 00:05:08 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Sat Oct 25 00:05:08 2025 -------------------
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85069896c3d/fastq, /tmp/RtmpNKqhpN/file1c85069896c3d/fastq/sample1.fq.gz, /tmp/RtmpNKqhpN/file1c85069896c3d/fastq/sample2.fq.gz, /tmp/RtmpNKqhpN/file1c85069896c3d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85069896c3d/sample1_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85069896c3d/sample2_matched_reads.fastq.gz, /tmp/RtmpNKqhpN/file1c85069896c3d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85069896c3d/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85069896c3d/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmpNKqhpN/file1c85069896c3d/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85069896c3d/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85069896c3d/sample1_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85069896c3d/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85069896c3d/sample2_realign2transcript.bam
/tmp/RtmpNKqhpN/file1c85069896c3d/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmpNKqhpN/file1c85069896c3d/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Sat Oct 25 00:05:27 2025 ----------
00:05:27 Sat Oct 25 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85069896c3d/sampleA_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample1_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85069896c3d/sample1_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample2_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85069896c3d/sample2_realign2transcript.bamdone
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample3_realign2transcript.bam...
parsing /tmp/RtmpNKqhpN/file1c85069896c3d/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmpNKqhpN/file1c85069896c3d/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
> 
> proc.time()
   user  system elapsed 
726.277  45.770 752.950