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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.4.2.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.2 (2025-10-31)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.4 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.4.2’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04.1) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     23.405  0.225  23.630
find_variants                20.438  0.063  19.883
blaze                         4.899 14.744  11.758
sc_long_multisample_pipeline  8.411  6.469   8.560
bulk_long_pipeline            2.355 11.297   2.504
sc_plot_genotype             10.797  1.021  10.658
MultiSampleSCPipeline        10.053  0.594  10.985
sc_DTU_analysis               7.104  2.071   6.902
plot_isoform_heatmap          7.111  0.094   7.204
create_sce_from_dir           3.630  2.585   3.744
* checking for unstated dependencies in ‘tests’ ... OK
* checking tests ...
  Running ‘testthat.R’
 OK
* checking for unstated dependencies in vignettes ... OK
* checking package vignettes ... OK
* checking re-building of vignette outputs ... OK
* checking PDF version of manual ... OK
* DONE

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

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

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

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

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Type 'contributors()' for more information and
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Type 'demo()' for some demos, 'help()' for on-line help, or
'help.start()' for an HTML browser interface to help.
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> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71640cd5cf6/config_file_1496854.json 
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71640cd5cf6/config_file_1496854.json 
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71640cd5cf6/config_file_1496854.json 
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71628ed0797/config_file_1496854.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/Rtmpl33SQi/file16d716df53c4b/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/Rtmpl33SQi/file16d7161c0a39e9/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/Rtmpl33SQi/file16d7161c0a39e9/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7169828244/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7169828244/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7169828244/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7169828244/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/Rtmpl33SQi/file16d716660e1018/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/Rtmpl33SQi/file16d7163af1b61b/config_file_1496854.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Mar 25 23:44:49 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpl33SQi/file16d7163af1b61b/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpl33SQi/file16d7163af1b61b/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpl33SQi/file16d7163af1b61b/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Mar 25 23:44:50 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:45:13 2026 -------------------
Realigning sample sample1 -> /tmp/Rtmpl33SQi/file16d7163af1b61b/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpl33SQi/file16d7163af1b61b/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpl33SQi/file16d7163af1b61b/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Mar 25 23:45:14 2026 ----------
2026-03-26T03:45:14.039702Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:45:14.040160Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163af1b61b/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:45:14.040171Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:45:14.040187Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:45:14.040242Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:45:14.040247Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:45:14.042032Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:45:14.042153Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2026-03-26T03:45:14.042175Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-03-26T03:45:14.042178Z  INFO oarfish::bulk: number of aligned reads : 96
2026-03-26T03:45:14.042180Z  INFO oarfish::bulk: number of unique alignments : 86
2026-03-26T03:45:14.042792Z  INFO oarfish: oarfish completed successfully.
2026-03-26T03:45:14.049546Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:45:14.049867Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163af1b61b/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:45:14.049874Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:45:14.049877Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:45:14.049928Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:45:14.049933Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:45:14.051557Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:45:14.051680Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2026-03-26T03:45:14.051704Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-03-26T03:45:14.051707Z  INFO oarfish::bulk: number of aligned reads : 95
2026-03-26T03:45:14.051720Z  INFO oarfish::bulk: number of unique alignments : 82
2026-03-26T03:45:14.052319Z  INFO oarfish: oarfish completed successfully.
2026-03-26T03:45:14.058930Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:45:14.059268Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163af1b61b/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:45:14.059327Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:45:14.059330Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:45:14.059381Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:45:14.059386Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:45:14.062170Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-03-26T03:45:14.062318Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2026-03-26T03:45:14.062347Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-03-26T03:45:14.062350Z  INFO oarfish::bulk: number of aligned reads : 179
2026-03-26T03:45:14.062352Z  INFO oarfish::bulk: number of unique alignments : 143
2026-03-26T03:45:14.063001Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71654618699/config_file_1496854.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Mar 25 23:45:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpl33SQi/file16d71654618699/sample1_align2genome.bam
sample2 ->/tmp/Rtmpl33SQi/file16d71654618699/sample2_align2genome.bam
sample3 ->/tmp/Rtmpl33SQi/file16d71654618699/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Mar 25 23:45:34 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:45:54 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpl33SQi/file16d71654618699/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpl33SQi/file16d71654618699/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpl33SQi/file16d71654618699/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:46:14 2026 ----------
2026-03-26T03:46:15.015376Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:46:15.015798Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71654618699/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:46:15.015808Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:46:15.015812Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:46:15.015888Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:46:15.015895Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:46:15.017510Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:46:15.017661Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2026-03-26T03:46:15.017686Z  INFO oarfish::bulk: Total number of alignment records : 116
2026-03-26T03:46:15.017689Z  INFO oarfish::bulk: number of aligned reads : 96
2026-03-26T03:46:15.017692Z  INFO oarfish::bulk: number of unique alignments : 86
2026-03-26T03:46:15.018290Z  INFO oarfish: oarfish completed successfully.
2026-03-26T03:46:15.030315Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:46:15.030672Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71654618699/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:46:15.030683Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:46:15.030700Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:46:15.030756Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:46:15.030761Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:46:15.032361Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:46:15.032485Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2026-03-26T03:46:15.032510Z  INFO oarfish::bulk: Total number of alignment records : 114
2026-03-26T03:46:15.032512Z  INFO oarfish::bulk: number of aligned reads : 95
2026-03-26T03:46:15.032514Z  INFO oarfish::bulk: number of unique alignments : 82
2026-03-26T03:46:15.033123Z  INFO oarfish: oarfish completed successfully.
2026-03-26T03:46:15.044159Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:46:15.044530Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71654618699/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:46:15.044540Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:46:15.044544Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:46:15.044596Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:46:15.044601Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:46:15.047361Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2026-03-26T03:46:15.047520Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2026-03-26T03:46:15.047549Z  INFO oarfish::bulk: Total number of alignment records : 239
2026-03-26T03:46:15.047552Z  INFO oarfish::bulk: number of aligned reads : 179
2026-03-26T03:46:15.047565Z  INFO oarfish::bulk: number of unique alignments : 143
2026-03-26T03:46:15.048237Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71624581dfd/config_file_1496854.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Mar 25 23:46:15 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpl33SQi/file16d71624581dfd/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpl33SQi/file16d71624581dfd/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpl33SQi/file16d71624581dfd/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Mar 25 23:46:16 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:46:34 2026 -------------------
Realigning sample sample1 -> /tmp/Rtmpl33SQi/file16d71624581dfd/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpl33SQi/file16d71624581dfd/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpl33SQi/file16d71624581dfd/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Mar 25 23:46:34 2026 ----------
23:46:34 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d716622f39aa/config_file_1496854.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Mar 25 23:46:36 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpl33SQi/file16d716622f39aa/sample1_align2genome.bam
sample2 ->/tmp/Rtmpl33SQi/file16d716622f39aa/sample2_align2genome.bam
sample3 ->/tmp/Rtmpl33SQi/file16d716622f39aa/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Mar 25 23:46:56 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:47:15 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpl33SQi/file16d716622f39aa/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpl33SQi/file16d716622f39aa/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpl33SQi/file16d716622f39aa/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:47:34 2026 ----------
23:47:34 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmpl33SQi/file16d71624581dfd/sample1_realign2transcript.bam', '/tmp/Rtmpl33SQi/file16d71624581dfd/sample2_realign2transcript.bam', '/tmp/Rtmpl33SQi/file16d71624581dfd/sample3_realign2transcript.bam'] /tmp/Rtmpl33SQi/file16d71624581dfd/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/Rtmpl33SQi/file16d71675b8a668/config_file_1496854.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Mar 25 23:47:35 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpl33SQi/file16d71675b8a668/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpl33SQi/file16d71675b8a668/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpl33SQi/file16d71675b8a668/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Mar 25 23:47:36 2026 -------------
Inputs:  ['/tmp/Rtmpl33SQi/file16d716622f39aa/sample1_realign2transcript.bam', '/tmp/Rtmpl33SQi/file16d716622f39aa/sample2_realign2transcript.bam', '/tmp/Rtmpl33SQi/file16d716622f39aa/sample3_realign2transcript.bam'] /tmp/Rtmpl33SQi/file16d716622f39aa/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 375, 'not_enough_coverage': 16, 'unmapped': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:47:36 2026 -------------------
Realigning sample sample1 -> /tmp/Rtmpl33SQi/file16d71675b8a668/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmpl33SQi/file16d71675b8a668/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmpl33SQi/file16d71675b8a668/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Wed Mar 25 23:47:37 2026 ----------
2026-03-26T03:47:37.736898Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:47:37.737416Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71675b8a668/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-03-26T03:47:37.737429Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:47:37.737432Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:47:37.737508Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:47:37.737516Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-26T03:47:37.740169Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:47:37.740320Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2026-03-26T03:47:37.740344Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-03-26T03:47:37.740347Z  INFO oarfish::bulk: number of aligned reads : 98
2026-03-26T03:47:37.740350Z  INFO oarfish::bulk: number of unique alignments : 86
2026-03-26T03:47:37.740964Z  INFO oarfish: oarfish completed successfully.
2026-03-26T03:47:37.748586Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:47:37.749107Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71675b8a668/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-03-26T03:47:37.749116Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:47:37.749120Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:47:37.749190Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:47:37.749197Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-26T03:47:37.751874Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:47:37.752023Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2026-03-26T03:47:37.752047Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-03-26T03:47:37.752049Z  INFO oarfish::bulk: number of aligned reads : 97
2026-03-26T03:47:37.752051Z  INFO oarfish::bulk: number of unique alignments : 79
2026-03-26T03:47:37.752659Z  INFO oarfish: oarfish completed successfully.
2026-03-26T03:47:37.759999Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:47:37.760383Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71675b8a668/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-03-26T03:47:37.760395Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:47:37.760398Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:47:37.760472Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:47:37.760478Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-26T03:47:37.764916Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:47:37.765085Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2026-03-26T03:47:37.765112Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-03-26T03:47:37.765115Z  INFO oarfish::bulk: number of aligned reads : 187
2026-03-26T03:47:37.765117Z  INFO oarfish::bulk: number of unique alignments : 140
2026-03-26T03:47:37.765827Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71641eedcfd/config_file_1496854.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Mar 25 23:47:37 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpl33SQi/file16d71641eedcfd/sample1_align2genome.bam
sample2 ->/tmp/Rtmpl33SQi/file16d71641eedcfd/sample2_align2genome.bam
sample3 ->/tmp/Rtmpl33SQi/file16d71641eedcfd/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Mar 25 23:47:57 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:47:57 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpl33SQi/file16d71641eedcfd/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpl33SQi/file16d71641eedcfd/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpl33SQi/file16d71641eedcfd/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:48:17 2026 ----------
2026-03-26T03:48:17.867428Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:48:17.867808Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71641eedcfd/sample1_realign2transcript.bam, contains 10 reference sequences.
2026-03-26T03:48:17.867818Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:48:17.867822Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:48:17.867891Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:48:17.867898Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-26T03:48:17.870538Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:48:17.870687Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2026-03-26T03:48:17.870710Z  INFO oarfish::bulk: Total number of alignment records : 125
2026-03-26T03:48:17.870714Z  INFO oarfish::bulk: number of aligned reads : 98
2026-03-26T03:48:17.870716Z  INFO oarfish::bulk: number of unique alignments : 86
2026-03-26T03:48:17.871345Z  INFO oarfish: oarfish completed successfully.
2026-03-26T03:48:17.882692Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:48:17.883067Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71641eedcfd/sample2_realign2transcript.bam, contains 10 reference sequences.
2026-03-26T03:48:17.883078Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:48:17.883081Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:48:17.883165Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:48:17.883172Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-26T03:48:17.885813Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:48:17.885953Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2026-03-26T03:48:17.885976Z  INFO oarfish::bulk: Total number of alignment records : 136
2026-03-26T03:48:17.885979Z  INFO oarfish::bulk: number of aligned reads : 97
2026-03-26T03:48:17.885981Z  INFO oarfish::bulk: number of unique alignments : 79
2026-03-26T03:48:17.886604Z  INFO oarfish: oarfish completed successfully.
2026-03-26T03:48:17.897723Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:48:17.898099Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71641eedcfd/sample3_realign2transcript.bam, contains 10 reference sequences.
2026-03-26T03:48:17.898106Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:48:17.898109Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:48:17.898177Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:48:17.898183Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-26T03:48:17.902460Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2026-03-26T03:48:17.902629Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2026-03-26T03:48:17.902658Z  INFO oarfish::bulk: Total number of alignment records : 272
2026-03-26T03:48:17.902660Z  INFO oarfish::bulk: number of aligned reads : 187
2026-03-26T03:48:17.902663Z  INFO oarfish::bulk: number of unique alignments : 140
2026-03-26T03:48:17.903388Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71614c01121/config_file_1496854.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Mar 25 23:48:18 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmpl33SQi/file16d71614c01121/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmpl33SQi/file16d71614c01121/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmpl33SQi/file16d71614c01121/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Mar 25 23:48:19 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:48:19 2026 -------------------
Realigning sample sample1 -> /tmp/Rtmpl33SQi/file16d71614c01121/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmpl33SQi/file16d71614c01121/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmpl33SQi/file16d71614c01121/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Mar 25 23:48:20 2026 ----------
23:48:20 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7166ae4ace3/config_file_1496854.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Wed Mar 25 23:48:21 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample1_align2genome.bam
sample2 ->/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample2_align2genome.bam
sample3 ->/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample3_align2genome.bam
-- Running step: isoform_identification @ Wed Mar 25 23:48:40 2026 -------------
Inputs:  ['/tmp/Rtmpl33SQi/file16d71614c01121/sample1_realign2transcript.bam', '/tmp/Rtmpl33SQi/file16d71614c01121/sample2_realign2transcript.bam', '/tmp/Rtmpl33SQi/file16d71614c01121/sample3_realign2transcript.bam'] /tmp/Rtmpl33SQi/file16d71614c01121/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:48:41 2026 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:49:02 2026 ----------
23:49:02 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7162166bab9/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:49:03 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7162166bab9/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 @ Wed Mar 25 23:49:03 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpl33SQi/file16d7162166bab9/matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162166bab9/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Mar 25 23:49:03 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:49:13 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7162166bab9/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7162166bab9/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpl33SQi/file16d7162166bab9/matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162166bab9/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Wed Mar 25 23:49:13 2026 ----------
2026-03-26T03:49:13.714080Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:49:13.714532Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7162166bab9/realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:49:13.714544Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:49:13.714548Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:49:13.714603Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:49:13.714611Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:49:13.721115Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7163e5dc979/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:49:14 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7163e5dc979/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 @ Wed Mar 25 23:49:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7163e5dc979/matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163e5dc979/align2genome.bam
-- Running step: isoform_identification @ Wed Mar 25 23:49:33 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:49:43 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163e5dc979/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163e5dc979/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7163e5dc979/matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163e5dc979/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:50:02 2026 ----------
2026-03-26T03:50:02.390042Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:50:02.390597Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163e5dc979/realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:50:02.390611Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:50:02.390615Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:50:02.390682Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:50:02.390688Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:50:02.398160Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71640268674/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:50:02 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d71640268674/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 @ Wed Mar 25 23:50:03 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpl33SQi/file16d71640268674/matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d71640268674/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Mar 25 23:50:03 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:50:13 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d71640268674/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d71640268674/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpl33SQi/file16d71640268674/matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d71640268674/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Wed Mar 25 23:50:13 2026 ----------
23:50:13 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample1_realign2transcript.bam', '/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample2_realign2transcript.bam', '/tmp/Rtmpl33SQi/file16d7166ae4ace3/sample3_realign2transcript.bam'] /tmp/Rtmpl33SQi/file16d7166ae4ace3/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/Rtmpl33SQi/file16d7163cdfa445/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:50:14 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7163cdfa445/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 @ Wed Mar 25 23:50:14 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7163cdfa445/matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163cdfa445/align2genome.bam
-- Running step: isoform_identification @ Wed Mar 25 23:50:32 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:50:42 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163cdfa445/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163cdfa445/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7163cdfa445/matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163cdfa445/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:51:01 2026 ----------
23:51:01 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71661b8f5f2/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:51:01 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d71661b8f5f2/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 @ Wed Mar 25 23:51:02 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpl33SQi/file16d71661b8f5f2/matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d71661b8f5f2/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Mar 25 23:51:02 2026 -------------
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:51:02 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d71661b8f5f2/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d71661b8f5f2/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpl33SQi/file16d71661b8f5f2/matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d71661b8f5f2/realign2transcript.bam
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Wed Mar 25 23:51:03 2026 ----------
2026-03-26T03:51:03.058441Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:51:03.058947Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d71661b8f5f2/realign2transcript.bam, contains 10 reference sequences.
2026-03-26T03:51:03.058955Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:51:03.058958Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:51:03.059024Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:51:03.059031Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-26T03:51:03.068520Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7164f68364b/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:51:03 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7164f68364b/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 @ Wed Mar 25 23:51:03 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7164f68364b/matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7164f68364b/align2genome.bam
-- Running step: isoform_identification @ Wed Mar 25 23:51:22 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:51:22 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7164f68364b/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7164f68364b/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7164f68364b/matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7164f68364b/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:51:42 2026 ----------
2026-03-26T03:51:42.402351Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:51:42.402886Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7164f68364b/realign2transcript.bam, contains 10 reference sequences.
2026-03-26T03:51:42.402896Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:51:42.402901Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:51:42.402983Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:51:42.402992Z  INFO oarfish: parsed reference information for 10 transcripts.
2026-03-26T03:51:42.412772Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7162ee6f20e/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:51:43 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7162ee6f20e/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 @ Wed Mar 25 23:51:43 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpl33SQi/file16d7162ee6f20e/matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162ee6f20e/align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Wed Mar 25 23:51:43 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:51:43 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7162ee6f20e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7162ee6f20e/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/Rtmpl33SQi/file16d7162ee6f20e/matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162ee6f20e/realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Wed Mar 25 23:51:44 2026 ----------
23:51:44 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d716549abf76/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: FALSE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:51:45 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d716549abf76/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 @ Wed Mar 25 23:51:45 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d716549abf76/matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d716549abf76/align2genome.bam
-- Running step: isoform_identification @ Wed Mar 25 23:52:04 2026 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:52:04 2026 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d716549abf76/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d716549abf76/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d716549abf76/matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d716549abf76/realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:52:22 2026 ----------
23:52:22 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7165dc5eb0a/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:52:24 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7165dc5eb0a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7165dc5eb0a/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7165dc5eb0a/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7165dc5eb0a/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/Rtmpl33SQi/file16d7165dc5eb0a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7165dc5eb0a/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/Rtmpl33SQi/file16d7165dc5eb0a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7165dc5eb0a/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/Rtmpl33SQi/file16d7165dc5eb0a/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7165dc5eb0a/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Wed Mar 25 23:52:24 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Mar 25 23:52:26 2026 ----------------
23:52:26 Wed Mar 25 2026 quantify genes 
Using BAM(s): '/tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_align2genome.bam', and
'/tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.31gene_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, 424301.38Read/s]
parsing /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.56gene_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, 1405597.86Read/s]
parsing /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.58gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1371225.32Read/s]
parsing /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.21gene_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, 750108.02Read/s]
-- Running step: isoform_identification @ Wed Mar 25 23:52:28 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:52:51 2026 -------------------
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7165dc5eb0a/fastq, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/fastq/sample1.fq.gz, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/fastq/sample2.fq.gz, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Mar 25 23:52:51 2026 ----------
2026-03-26T03:52:51.943011Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:52:51.943473Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:52:51.943485Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:52:51.943488Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:52:51.943538Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:52:51.943544Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:52:51.949215Z  INFO oarfish::single_cell: Processed 100 cells.
2026-03-26T03:52:52.244004Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:52:52.244498Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:52:52.244510Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:52:52.244513Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:52:52.244573Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:52:52.244579Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:52:52.557853Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:52:52.558224Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:52:52.558232Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:52:52.558235Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:52:52.558298Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:52:52.558307Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:52:52.859482Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:52:52.859965Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7165dc5eb0a/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:52:52.859972Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:52:52.859975Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:52:52.860031Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:52:52.860037Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7163f1b0f31/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:52:53 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7163f1b0f31/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7163f1b0f31/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7163f1b0f31/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7163f1b0f31/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/Rtmpl33SQi/file16d7163f1b0f31/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7163f1b0f31/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/Rtmpl33SQi/file16d7163f1b0f31/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7163f1b0f31/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/Rtmpl33SQi/file16d7163f1b0f31/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7163f1b0f31/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Wed Mar 25 23:52:54 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_align2genome.bam
/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_align2genome.bam
/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_align2genome.bam
/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Mar 25 23:53:13 2026 ----------------
23:53:13 Wed Mar 25 2026 quantify genes 
Using BAM(s): '/tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_align2genome.bam', and
'/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_align2genome.bam'
parsing /tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.97gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 416118.10Read/s]
parsing /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.84gene_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, 1495188.93Read/s]
parsing /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.00gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1307125.41Read/s]
parsing /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.05gene_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, 785332.53Read/s]
-- Running step: isoform_identification @ Wed Mar 25 23:53:14 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:53:37 2026 -------------------
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163f1b0f31/fastq, /tmp/Rtmpl33SQi/file16d7163f1b0f31/fastq/sample1.fq.gz, /tmp/Rtmpl33SQi/file16d7163f1b0f31/fastq/sample2.fq.gz, /tmp/Rtmpl33SQi/file16d7163f1b0f31/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:53:57 2026 ----------
2026-03-26T03:53:57.672023Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:53:57.672554Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163f1b0f31/sampleA_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:53:57.672567Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:53:57.672571Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:53:57.672630Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:53:57.672636Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:53:57.678872Z  INFO oarfish::single_cell: Processed 100 cells.
2026-03-26T03:53:58.051038Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:53:58.051620Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample1_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:53:58.051632Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:53:58.051636Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:53:58.051688Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:53:58.051694Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:53:58.422789Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:53:58.423300Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample2_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:53:58.423314Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:53:58.423317Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:53:58.423392Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:53:58.423397Z  INFO oarfish: parsed reference information for 5 transcripts.
2026-03-26T03:53:58.742415Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:53:58.742959Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163f1b0f31/sample3_realign2transcript.bam, contains 5 reference sequences.
2026-03-26T03:53:58.742968Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:53:58.742972Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:53:58.743025Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:53:58.743031Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7162f64beb8/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:53:59 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7162f64beb8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7162f64beb8/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7162f64beb8/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7162f64beb8/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Wed Mar 25 23:54:00 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpl33SQi/file16d7162f64beb8/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/sample1_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/sample2_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/sample3_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Mar 25 23:54:01 2026 ----------------
23:54:01 Wed Mar 25 2026 quantify genes 
Using BAM(s): '/tmp/Rtmpl33SQi/file16d7162f64beb8/sampleA_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7162f64beb8/sample1_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7162f64beb8/sample2_align2genome.bam', and
'/tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_align2genome.bam'
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/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, 459216.96Read/s]
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/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, 1331694.18Read/s]
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sample2_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1332201.75Read/s]
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.97gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 732245.81Read/s]
-- Running step: isoform_identification @ Wed Mar 25 23:54:02 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:54:26 2026 -------------------
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7162f64beb8/fastq, /tmp/Rtmpl33SQi/file16d7162f64beb8/fastq/sample1.fq.gz, /tmp/Rtmpl33SQi/file16d7162f64beb8/fastq/sample2.fq.gz, /tmp/Rtmpl33SQi/file16d7162f64beb8/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7162f64beb8/sampleA_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7162f64beb8/sample1_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7162f64beb8/sample2_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7162f64beb8/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7162f64beb8/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7162f64beb8/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpl33SQi/file16d7162f64beb8/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162f64beb8/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/Rtmpl33SQi/file16d7162f64beb8/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Mar 25 23:54:27 2026 ----------
23:54:27 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d7162f64beb8/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sample1_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d7162f64beb8/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sample2_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d7162f64beb8/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d7162f64beb8/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d716440c2213/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:54:29 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d716440c2213/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d716440c2213/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d716440c2213/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d716440c2213/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/Rtmpl33SQi/file16d716440c2213/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d716440c2213/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/Rtmpl33SQi/file16d716440c2213/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d716440c2213/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/Rtmpl33SQi/file16d716440c2213/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d716440c2213/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Wed Mar 25 23:54:29 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d716440c2213/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d716440c2213/sampleA_align2genome.bam
/tmp/Rtmpl33SQi/file16d716440c2213/sample1_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d716440c2213/sample1_align2genome.bam
/tmp/Rtmpl33SQi/file16d716440c2213/sample2_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d716440c2213/sample2_align2genome.bam
/tmp/Rtmpl33SQi/file16d716440c2213/sample3_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d716440c2213/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Mar 25 23:54:50 2026 ----------------
23:54:50 Wed Mar 25 2026 quantify genes 
Using BAM(s): '/tmp/Rtmpl33SQi/file16d716440c2213/sampleA_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d716440c2213/sample1_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d716440c2213/sample2_align2genome.bam', and
'/tmp/Rtmpl33SQi/file16d716440c2213/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/Rtmpl33SQi/file16d716440c2213/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.67gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 434336.84Read/s]
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.17gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1476659.63Read/s]
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.70gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1233618.82Read/s]
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.71gene_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, 783747.66Read/s]
-- Running step: isoform_identification @ Wed Mar 25 23:54:51 2026 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Wed Mar 25 23:55:13 2026 -------------------
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d716440c2213/fastq, /tmp/Rtmpl33SQi/file16d716440c2213/fastq/sample1.fq.gz, /tmp/Rtmpl33SQi/file16d716440c2213/fastq/sample2.fq.gz, /tmp/Rtmpl33SQi/file16d716440c2213/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d716440c2213/sampleA_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d716440c2213/sample1_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d716440c2213/sample2_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d716440c2213/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d716440c2213/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d716440c2213/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d716440c2213/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d716440c2213/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d716440c2213/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d716440c2213/sampleA_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d716440c2213/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d716440c2213/sample1_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d716440c2213/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d716440c2213/sample2_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d716440c2213/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d716440c2213/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:55:33 2026 ----------
23:55:33 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d716440c2213/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample1_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d716440c2213/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample2_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d716440c2213/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample3_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d716440c2213/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d716440c2213/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d716942acc0/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:55:35 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d716942acc0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d716942acc0/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d716942acc0/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d716942acc0/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/Rtmpl33SQi/file16d716942acc0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d716942acc0/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/Rtmpl33SQi/file16d716942acc0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d716942acc0/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/Rtmpl33SQi/file16d716942acc0/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d716942acc0/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Wed Mar 25 23:55:35 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpl33SQi/file16d716942acc0/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d716942acc0/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpl33SQi/file16d716942acc0/sample1_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d716942acc0/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpl33SQi/file16d716942acc0/sample2_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d716942acc0/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmpl33SQi/file16d716942acc0/sample3_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d716942acc0/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Mar 25 23:55:37 2026 ----------------
23:55:37 Wed Mar 25 2026 quantify genes 
Using BAM(s): '/tmp/Rtmpl33SQi/file16d716942acc0/sampleA_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d716942acc0/sample1_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d716942acc0/sample2_align2genome.bam', and
'/tmp/Rtmpl33SQi/file16d716942acc0/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/Rtmpl33SQi/file16d716942acc0/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.26gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 472714.81Read/s]
parsing /tmp/Rtmpl33SQi/file16d716942acc0/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1444319.56Read/s]
parsing /tmp/Rtmpl33SQi/file16d716942acc0/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.86gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1312524.72Read/s]
parsing /tmp/Rtmpl33SQi/file16d716942acc0/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.11gene_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, 780771.41Read/s]
-- Running step: isoform_identification @ Wed Mar 25 23:55:38 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:55:38 2026 -------------------
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d716942acc0/fastq, /tmp/Rtmpl33SQi/file16d716942acc0/fastq/sample1.fq.gz, /tmp/Rtmpl33SQi/file16d716942acc0/fastq/sample2.fq.gz, /tmp/Rtmpl33SQi/file16d716942acc0/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d716942acc0/sampleA_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d716942acc0/sample1_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d716942acc0/sample2_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d716942acc0/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d716942acc0/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d716942acc0/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d716942acc0/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d716942acc0/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpl33SQi/file16d716942acc0/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d716942acc0/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpl33SQi/file16d716942acc0/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d716942acc0/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpl33SQi/file16d716942acc0/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d716942acc0/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmpl33SQi/file16d716942acc0/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d716942acc0/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Wed Mar 25 23:55:40 2026 ----------
2026-03-26T03:55:40.609296Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:55:40.609783Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d716942acc0/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-03-26T03:55:40.609792Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:55:40.609795Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:55:40.609884Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:55:40.609892Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-26T03:55:40.621731Z  INFO oarfish::single_cell: Processed 100 cells.
2026-03-26T03:55:41.147078Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:55:41.147701Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d716942acc0/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-03-26T03:55:41.147714Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:55:41.147718Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:55:41.147815Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:55:41.147823Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-26T03:55:41.705998Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:55:41.706537Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d716942acc0/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-03-26T03:55:41.706549Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:55:41.706552Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:55:41.706636Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:55:41.706656Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-26T03:55:42.224862Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:55:42.225332Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d716942acc0/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-03-26T03:55:42.225344Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:55:42.225346Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:55:42.225430Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:55:42.225437Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7163f98a8c5/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:55:42 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7163f98a8c5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7163f98a8c5/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7163f98a8c5/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7163f98a8c5/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/Rtmpl33SQi/file16d7163f98a8c5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7163f98a8c5/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/Rtmpl33SQi/file16d7163f98a8c5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7163f98a8c5/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/Rtmpl33SQi/file16d7163f98a8c5/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7163f98a8c5/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Wed Mar 25 23:55:43 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_align2genome.bam
/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_align2genome.bam
/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_align2genome.bam
/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Mar 25 23:56:03 2026 ----------------
23:56:03 Wed Mar 25 2026 quantify genes 
Using BAM(s): '/tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_align2genome.bam', and
'/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_align2genome.bam'
parsing /tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.57gene_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, 342291.57Read/s]
parsing /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.14gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1180430.03Read/s]
parsing /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.16gene_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, 1200980.41Read/s]
parsing /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.00gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 747008.62Read/s]
-- Running step: isoform_identification @ Wed Mar 25 23:56:04 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:56:05 2026 -------------------
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163f98a8c5/fastq, /tmp/Rtmpl33SQi/file16d7163f98a8c5/fastq/sample1.fq.gz, /tmp/Rtmpl33SQi/file16d7163f98a8c5/fastq/sample2.fq.gz, /tmp/Rtmpl33SQi/file16d7163f98a8c5/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:56:25 2026 ----------
2026-03-26T03:56:25.459320Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:56:25.459822Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163f98a8c5/sampleA_realign2transcript.bam, contains 14 reference sequences.
2026-03-26T03:56:25.459832Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:56:25.459836Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:56:25.459916Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:56:25.459925Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-26T03:56:25.471692Z  INFO oarfish::single_cell: Processed 100 cells.
2026-03-26T03:56:26.128933Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:56:26.129348Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample1_realign2transcript.bam, contains 14 reference sequences.
2026-03-26T03:56:26.129359Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:56:26.129363Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:56:26.129444Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:56:26.129452Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-26T03:56:26.696527Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:56:26.696912Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample2_realign2transcript.bam, contains 14 reference sequences.
2026-03-26T03:56:26.696922Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:56:26.696926Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:56:26.697009Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:56:26.697018Z  INFO oarfish: parsed reference information for 14 transcripts.
2026-03-26T03:56:27.248865Z  INFO oarfish: setting user-provided filter parameters.
2026-03-26T03:56:27.249413Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmpl33SQi/file16d7163f98a8c5/sample3_realign2transcript.bam, contains 14 reference sequences.
2026-03-26T03:56:27.249426Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2026-03-26T03:56:27.249429Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2026-03-26T03:56:27.249511Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2026-03-26T03:56:27.249519Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d71675576bdf/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:56:28 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d71675576bdf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d71675576bdf/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d71675576bdf/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d71675576bdf/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Wed Mar 25 23:56:28 2026 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/sample1_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/sample2_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/sample3_matched_reads.fastq.gz -> /tmp/Rtmpl33SQi/file16d71675576bdf/sample3_align2genome.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Wed Mar 25 23:56:30 2026 ----------------
23:56:30 Wed Mar 25 2026 quantify genes 
Using BAM(s): '/tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d71675576bdf/sample1_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d71675576bdf/sample2_align2genome.bam', and
'/tmp/Rtmpl33SQi/file16d71675576bdf/sample3_align2genome.bam'
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.80gene_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, 406487.83Read/s]
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.66gene_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, 1357379.94Read/s]
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.43gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1349692.37Read/s]
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.86gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 791736.64Read/s]
-- Running step: isoform_identification @ Wed Mar 25 23:56:31 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:56:31 2026 -------------------
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d71675576bdf/fastq, /tmp/Rtmpl33SQi/file16d71675576bdf/fastq/sample1.fq.gz, /tmp/Rtmpl33SQi/file16d71675576bdf/fastq/sample2.fq.gz, /tmp/Rtmpl33SQi/file16d71675576bdf/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d71675576bdf/sample1_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d71675576bdf/sample2_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d71675576bdf/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d71675576bdf/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d71675576bdf/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d71675576bdf/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d71675576bdf/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/Rtmpl33SQi/file16d71675576bdf/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmpl33SQi/file16d71675576bdf/sample3_realign2transcript.bam
Your fastq file appears to have tags, but you did not provide the -y option to minimap2 to include the tags in the output.
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Wed Mar 25 23:56:32 2026 ----------
23:56:32 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d71675576bdf/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample1_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d71675576bdf/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample2_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d71675576bdf/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample3_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d71675576bdf/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d71675576bdf/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmpl33SQi/file16d7166b7bc439/config_file_1496854.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Wed Mar 25 23:56:35 2026 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/Rtmpl33SQi/file16d7166b7bc439/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7166b7bc439/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7166b7bc439/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmpl33SQi/file16d7166b7bc439/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/Rtmpl33SQi/file16d7166b7bc439/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7166b7bc439/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/Rtmpl33SQi/file16d7166b7bc439/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7166b7bc439/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/Rtmpl33SQi/file16d7166b7bc439/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmpl33SQi/file16d7166b7bc439/fastq/sample3.fq.gz
Searching for barcodes...
Number of reads processed: 193
Number of reads where at least one barcode was found: 181
Number of reads with exactly one barcode match: 179
Number of chimera reads: 0
All done!
Reads	Barcodes
7	1
6	1
5	1
4	7
3	10
2	27
1	53
-- Running step: genome_alignment @ Wed Mar 25 23:56:36 2026 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_align2genome.bam
/tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_align2genome.bam
/tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_align2genome.bam
/tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_matched_reads.fastq.gz ->/tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_align2genome.bam
-- Running step: gene_quantification @ Wed Mar 25 23:56:55 2026 ----------------
23:56:55 Wed Mar 25 2026 quantify genes 
Using BAM(s): '/tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_align2genome.bam',
'/tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_align2genome.bam', and
'/tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.55gene_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, 311742.18Read/s]
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.58gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1264410.95Read/s]
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.24gene_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, 1235654.02Read/s]
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 740886.03Read/s]
-- Running step: isoform_identification @ Wed Mar 25 23:56:56 2026 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Wed Mar 25 23:56:56 2026 -------------------
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7166b7bc439/fastq, /tmp/Rtmpl33SQi/file16d7166b7bc439/fastq/sample1.fq.gz, /tmp/Rtmpl33SQi/file16d7166b7bc439/fastq/sample2.fq.gz, /tmp/Rtmpl33SQi/file16d7166b7bc439/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_matched_reads.fastq.gz, /tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_realign2transcript.bam
/tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Wed Mar 25 23:57:16 2026 ----------
23:57:16 Wed Mar 25 2026 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d7166b7bc439/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d7166b7bc439/sample1_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d7166b7bc439/sample2_realign2transcript.bamdone
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_realign2transcript.bam...
parsing /tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmpl33SQi/file16d7166b7bc439/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 182 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 182 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
> 
> proc.time()
   user  system elapsed 
738.957  41.876 768.980