##############################################################################
##############################################################################
###
### 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_2.4.1.tar.gz
###
##############################################################################
##############################################################################


* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 Patched (2025-08-23 r88802)
* using platform: x86_64-pc-linux-gnu
* R was compiled by
    gcc (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
    GNU Fortran (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0
* running under: Ubuntu 24.04.3 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.4.1’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 47 non-default packages.
Importing from so many packages makes the package vulnerable to any of
them becoming unavailable.  Move as many as possible to Suggests and
use conditionally.
* checking if this is a source package ... OK
* checking if there is a namespace ... OK
* checking for hidden files and directories ... NOTE
Found the following hidden files and directories:
  .dockerignore
These were most likely included in error. See section ‘Package
structure’ in the ‘Writing R Extensions’ manual.
* checking for portable file names ... OK
* checking for sufficient/correct file permissions ... OK
* checking whether package ‘FLAMES’ can be installed ... NOTE
Found the following notes/warnings:
  Non-staged installation was used
See ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00install.out’ for details.
* used C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
* checking C++ specification ... OK
* checking installed package size ... INFO
  installed size is  6.0Mb
  sub-directories of 1Mb or more:
    bin    1.1Mb
    data   1.8Mb
    libs   1.5Mb
* checking package directory ... OK
* checking ‘build’ directory ... OK
* checking DESCRIPTION meta-information ... OK
* checking top-level files ... OK
* checking for left-over files ... OK
* checking index information ... OK
* checking package subdirectories ... OK
* checking code files for non-ASCII characters ... OK
* checking R files for syntax errors ... OK
* checking whether the package can be loaded ... OK
* checking whether the package can be loaded with stated dependencies ... OK
* checking whether the package can be unloaded cleanly ... OK
* checking whether the namespace can be loaded with stated dependencies ... OK
* checking whether the namespace can be unloaded cleanly ... OK
* checking loading without being on the library search path ... OK
* checking dependencies in R code ... NOTE
There are ::: calls to the package's namespace in its code. A package
  almost never needs to use ::: for its own objects:
  'blaze' 'find_barcode' 'gene_quantification' 'isoform_identification'
  'minimap2_align' 'transcript_quantification'
* checking S3 generic/method consistency ... OK
* checking replacement functions ... OK
* checking foreign function calls ... OK
* checking R code for possible problems ... NOTE
BulkPipeline: no visible global function definition for ‘new’
BulkPipeline: no visible global function definition for ‘setNames’
MultiSampleSCPipeline: no visible global function definition for ‘new’
MultiSampleSCPipeline: no visible global function definition for
  ‘setNames’
SingleCellPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for
  ‘setNames’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: no visible global function definition for ‘as’
add_gene_counts: no visible global function definition for ‘as’
cache_dir: no visible global function definition for ‘packageVersion’
chisq_test_by_gene: no visible global function definition for
  ‘chisq.test’
create_sce_from_dir: no visible global function definition for
  ‘setNames’
create_spe: no visible binding for global variable ‘barcode’
create_spe: no visible binding for global variable ‘in_tissue’
download_oarfish: no visible global function definition for
  ‘download.file’
download_oarfish: no visible global function definition for ‘unzip’
filter_coverage: no visible global function definition for
  ‘starts_with’
filter_coverage: no visible binding for global variable ‘filter_res’
find_variants: no visible global function definition for ‘setNames’
find_variants_grange: no visible binding for global variable
  ‘which_label’
find_variants_grange: no visible binding for global variable
  ‘nucleotide’
find_variants_grange: no visible binding for global variable ‘pos’
find_variants_grange: no visible binding for global variable ‘count’
find_variants_grange: no visible binding for global variable
  ‘counts_no_ins’
find_variants_grange: no visible binding for global variable ‘ref’
generate_sc_sce: no visible binding for global variable ‘FSM_match’
get_coverage: no visible binding for global variable ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘Freq’
homopolymer_pct : <anonymous>: no visible binding for global variable
  ‘pct’
plot_coverage: no visible binding for global variable ‘tr_length’
plot_coverage: no visible binding for global variable ‘read_counts’
plot_coverage: no visible binding for global variable ‘total_counts’
plot_coverage: no visible binding for global variable ‘cumpct’
plot_coverage: no visible binding for global variable ‘length_bin’
plot_coverage: no visible binding for global variable ‘min_length’
plot_coverage: no visible binding for global variable ‘max_length’
plot_coverage: no visible global function definition for ‘head’
plot_coverage: no visible binding for global variable ‘transcript’
plot_demultiplex_raw: no visible binding for global variable ‘Sample’
plot_demultiplex_raw: no visible binding for global variable
  ‘CellBarcode’
plot_demultiplex_raw: no visible binding for global variable ‘UMI’
plot_demultiplex_raw: no visible binding for global variable
  ‘UMI_count’
plot_demultiplex_raw: no visible binding for global variable
  ‘barcode_rank’
plot_demultiplex_raw: no visible binding for global variable
  ‘FlankEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘n_reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘BarcodeEditDist’
plot_demultiplex_raw: no visible binding for global variable ‘total
  reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘demultiplexed reads’
plot_demultiplex_raw: no visible binding for global variable ‘single
  match reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘undemultiplexted reads’
plot_demultiplex_raw: no visible binding for global variable
  ‘multi-matching reads’
plot_demultiplex_raw: no visible binding for global variable ‘Type’
plot_demultiplex_raw: no visible binding for global variable ‘Reads’
plot_demultiplex_raw: no visible binding for global variable ‘input’
plot_demultiplex_raw: no visible binding for global variable ‘output’
plot_demultiplex_raw: no visible binding for global variable
  ‘read1_with_adapter’
plot_demultiplex_raw: no visible binding for global variable ‘Count’
plot_flagstat: no visible global function definition for ‘everything’
plot_flagstat: no visible binding for global variable ‘name’
plot_flagstat: no visible binding for global variable ‘value’
plot_isoform_reduced_dim: no visible binding for global variable ‘x’
plot_isoform_reduced_dim: no visible binding for global variable ‘y’
plot_isoform_reduced_dim: no visible binding for global variable ‘expr’
plot_spatial: no visible binding for global variable ‘imageX’
plot_spatial: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘imageX’
plot_spatial_feature: no visible binding for global variable ‘imageY’
plot_spatial_feature: no visible binding for global variable ‘x’
plot_spatial_feature: no visible binding for global variable ‘y’
plot_spatial_feature: no visible global function definition for
  ‘scale_alpha_continuous’
plot_spatial_feature: no visible global function definition for
  ‘scale_colour_gradient’
plot_spatial_isoform: no visible global function definition for ‘head’
plot_spatial_pie: no visible global function definition for ‘setNames’
plot_spatial_pie: no visible global function definition for ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_gene_entropy: no visible global function definition for ‘as’
sc_genotype: no visible binding for global variable ‘allele’
sc_genotype: no visible binding for global variable ‘allele_count’
sc_genotype: no visible binding for global variable ‘barcode’
sc_genotype: no visible binding for global variable ‘pct’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_plot_genotype: no visible global function definition for ‘setNames’
sc_plot_genotype: no visible binding for global variable ‘barcode’
sc_plot_genotype: no visible binding for global variable ‘genotype’
sc_plot_genotype: no visible binding for global variable ‘x’
sc_plot_genotype: no visible binding for global variable ‘y’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible global
  function definition for ‘setNames’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline : <anonymous>: no
  visible global function definition for ‘setNames’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible global
  function definition for ‘setNames’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘genome_bam’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘threads’
genome_alignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘step’
plot_durations,FLAMES.Pipeline: no visible binding for global variable
  ‘duration’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘j’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_assembly’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘transcriptome_bam’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘minimap2’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘samtools’
read_realignment_raw,FLAMES.Pipeline: no visible binding for global
  variable ‘outdir’
resume_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
run_FLAMES,FLAMES.Pipeline : <anonymous>: no visible global function
  definition for ‘capture.output’
Undefined global functions or variables:
  BarcodeEditDist CellBarcode Count FSM_match FlankEditDist Freq Reads
  Sample Type UMI UMI_count adj.p.value allele allele_count as
  bam_index barcode barcode_rank barcodes_file capture.output
  cell_total_reads chisq.test count counts_no_ins cumpct demultiplexed
  reads demultiplexed_fastq download.file duration everything
  expect_cell_number expr fastq filter_res genome_bam genotype head
  imageX imageY in_tissue input j length_bin max_length min_length
  minimap2 multi-matching reads mutation_index n_reads na.omit name new
  nucleotide outdir output p.value packageVersion pct pos
  read1_with_adapter read_counts ref samtools scale_alpha_continuous
  scale_colour_gradient setNames single match reads starts_with step
  test threads total total reads total_counts tr_length transcript
  transcriptome_assembly transcriptome_bam undemultiplexted reads unzip
  value which_label x y
Consider adding
  importFrom("base", "match", "single")
  importFrom("methods", "as", "new")
  importFrom("stats", "chisq.test", "na.omit", "setNames", "step")
  importFrom("utils", "capture.output", "download.file", "head",
             "packageVersion", "unzip")
to your NAMESPACE file (and ensure that your DESCRIPTION Imports field
contains 'methods').
* checking Rd files ... OK
* checking Rd metadata ... OK
* checking Rd cross-references ... OK
* checking for missing documentation entries ... OK
* checking for code/documentation mismatches ... OK
* checking Rd \usage sections ... OK
* checking Rd contents ... OK
* checking for unstated dependencies in examples ... OK
* checking contents of ‘data’ directory ... OK
* checking data for non-ASCII characters ... OK
* checking data for ASCII and uncompressed saves ... OK
* checking line endings in shell scripts ... OK
* checking line endings in C/C++/Fortran sources/headers ... OK
* checking line endings in Makefiles ... OK
* checking compilation flags in Makevars ... OK
* checking for GNU extensions in Makefiles ... INFO
GNU make is a SystemRequirements.
* checking for portable use of $(BLAS_LIBS) and $(LAPACK_LIBS) ... OK
* checking use of PKG_*FLAGS in Makefiles ... OK
* checking include directives in Makefiles ... OK
* checking compiled code ... NOTE
Note: information on .o files is not available
File ‘/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs/FLAMES.so’:
  Found ‘abort’, possibly from ‘abort’ (C)
  Found ‘exit’, possibly from ‘exit’ (C)
  Found ‘stderr’, possibly from ‘stderr’ (C)
  Found ‘stdout’, possibly from ‘stdout’ (C)

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

See ‘Writing portable packages’ in the ‘Writing R Extensions’ manual.
* checking files in ‘vignettes’ ... OK
* checking examples ... OK
Examples with CPU (user + system) or elapsed time > 5s
                               user system elapsed
plot_isoform_reduced_dim     25.597  0.160  25.758
find_variants                20.814  0.068  20.272
blaze                         4.366 15.715  11.783
sc_long_multisample_pipeline  8.267  6.703   8.530
bulk_long_pipeline            2.450 11.620   2.517
sc_plot_genotype             12.046  0.937  11.829
MultiSampleSCPipeline        10.304  0.538  11.256
sc_DTU_analysis               7.251  1.880   7.022
plot_isoform_heatmap          7.441  0.131   7.571
create_sce_from_dir           3.693  2.110   3.827
* 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.

##############################################################################
##############################################################################
###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
##############################################################################


* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.4.1’
** using non-staged installation via StagedInstall field
** libs
using C++ compiler: ‘g++ (Ubuntu 13.3.0-6ubuntu2~24.04) 13.3.0’
using C++17
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppExports.cpp -o RcppExports.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c RcppFunctions.cpp -o RcppFunctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/BamRecord.cpp -o classes/BamRecord.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GFFRecord.cpp -o classes/GFFRecord.o
In file included from classes/GFFRecord.cpp:8:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/GeneAnnotationParser.cpp -o classes/GeneAnnotationParser.o
In file included from classes/GeneAnnotationParser.cpp:15:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/Isoforms.cpp -o classes/Isoforms.o
In file included from classes/Isoforms.cpp:16:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::update_all_splice()’:
classes/Isoforms.cpp:233:35: warning: comparison of integer expressions of different signedness: ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  233 |                 if (blocks.size() >= (int)this->Min_sup_cnt) {
      |                     ~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::filter_TSS_TES(std::ofstream&, DoubleJunctions, float)’:
classes/Isoforms.cpp:368:33: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  368 |         if ((left_counts.size() < (int)this->Min_sup_cnt) ||
      |              ~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:369:38: warning: comparison of integer expressions of different signedness: ‘std::unordered_map<int, int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  369 |                 (right_counts.size() < (int)this->Min_sup_cnt)) {
      |                  ~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp: In member function ‘void Isoforms::match_known_annotation(const std::unordered_map<std::__cxx11::basic_string<char>, Junctions>&, const std::unordered_map<std::__cxx11::basic_string<char>, Pos>&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&, GeneBlocks, std::unordered_map<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> >)’:
classes/Isoforms.cpp:662:51: warning: comparison of integer expressions of different signedness: ‘int’ and ‘const long unsigned int’ [-Wsign-compare]
  662 |                                 for (int j = 0; j < std::min(raw_iso_key.size() - 1, junction.junctions.size()); j++) {
      |                                                 ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:713:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  713 |                         for (int i = 0; i < new_exons.size(); ++i) {
      |                                         ~~^~~~~~~~~~~~~~~~~~
classes/Isoforms.cpp:725:46: warning: comparisons like ‘X<=Y<=Z’ do not have their mathematical meaning [-Wparentheses]
  725 |                                 } else if (0 < i < raw_iso_key.size() - 1) { // a site from the middle somewhere
      |                                            ~~^~~
classes/Isoforms.cpp: In member function ‘std::string Isoforms::isoform_to_gff3(float)’:
classes/Isoforms.cpp:1140:43: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
 1140 |                         for (int i = 0; i < exons.size(); i+=2) {
      |                                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c classes/junctions.cpp -o classes/junctions.o
In file included from classes/junctions.cpp:12:
classes/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
classes/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
classes/junctions.cpp: In function ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> > get_gene_flat(const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::__cxx11::basic_string<char> > >&, const std::unordered_map<std::__cxx11::basic_string<char>, std::vector<StartEndPair> >&)’:
classes/junctions.cpp:166:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<StartEndPair>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  166 |         for (int i = 1; i < exons.size(); i++) {
      |                         ~~^~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/find_isoform.cpp -o main-functions/find_isoform.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/flexiplex.cpp -o main-functions/flexiplex.o
main-functions/flexiplex.cpp: In function ‘unsigned int edit_distance(const std::string&, const std::string&, unsigned int&, int)’:
main-functions/flexiplex.cpp:123:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  123 |       if (min_value <= max_editd)
      |           ~~~~~~~~~~^~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::string get_umi(const std::string&, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&, const std::vector<int>&, int, int, bool, int, int)’:
main-functions/flexiplex.cpp:164:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  164 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:186:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  186 |     if (read_to_subpatterns.size() > umi_index + 1) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Barcode get_barcode(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:299:19: warning: comparison of integer expressions of different signedness: ‘int’ and ‘__gnu_cxx::__alloc_traits<std::allocator<long unsigned int>, long unsigned int>::value_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  299 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:358:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  358 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:360:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  360 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:361:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  361 |                editDistance <= barcode_max_editd) { // if best so far, update
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘std::vector<Barcode> big_barcode_search(const std::string&, const std::unordered_set<std::__cxx11::basic_string<char> >&, int, int, const std::vector<std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > >&)’:
main-functions/flexiplex.cpp:400:29: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  400 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void write_bgzfstring(BGZF*, const std::string&)’:
main-functions/flexiplex.cpp:423:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  423 |   if (bytes_written != str.size()) {
      |       ~~~~~~~~~~~~~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, BGZF*)’:
main-functions/flexiplex.cpp:438:28: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  438 |         (barcode.flank_end == std::string::npos ? "True" : "False") + "\n";
      |          ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, BGZF*, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:475:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  475 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:490:32: warning: comparison of integer expressions of different signedness: ‘const int’ and ‘const std::__cxx11::basic_string<char>::size_type’ {aka ‘const long unsigned int’} [-Wsign-compare]
  490 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:495:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  495 |     for (int f = 0; f < vec_bc.size(); f++) {
      |                     ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘Rcpp::IntegerVector flexiplex_cpp(Rcpp::StringVector, Rcpp::String, bool, int, int, Rcpp::StringVector, Rcpp::String, Rcpp::String, Rcpp::String, bool, int)’:
main-functions/flexiplex.cpp:754:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  754 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:759:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  759 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:761:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  761 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:763:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  763 |           for (int b = 0; b < sr_v[t][r].vec_bc_rev.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/get_transcript_seq.cpp -o main-functions/get_transcript_seq.o
main-functions/get_transcript_seq.cpp: In function ‘void write_fa(const std::string&, const std::string&, const std::string&, int)’:
main-functions/get_transcript_seq.cpp:87:14: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   87 |     while (i < seq.size()) {
      |            ~~^~~~~~~~~~~~
main-functions/get_transcript_seq.cpp:88:26: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
   88 |         if (i + wrap_len > seq.size()) {
      |             ~~~~~~~~~~~~~^~~~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/group_bam2isoform.cpp -o main-functions/group_bam2isoform.o
In file included from main-functions/group_bam2isoform.cpp:18:
main-functions/../utility/utility.h: In function ‘std::pair<std::__cxx11::basic_string<char>, std::__cxx11::basic_string<char> > parseSpace(const std::string&)’:
main-functions/../utility/utility.h:255:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  255 |         for (int i = 0; i < s.size(); i++) {
      |                         ~~^~~~~~~~~~
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c main-functions/pileup_readid.cpp -o main-functions/pileup_readid.o
main-functions/pileup_readid.cpp: In function ‘Rcpp::NumericMatrix variant_count_matrix_cpp(Rcpp::String, Rcpp::String, int, bool, bool)’:
main-functions/pileup_readid.cpp:268:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<char>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  268 |   for (int i = 0; i < BASES.size(); i++) {
      |                   ~~^~~~~~~~~~~~~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, unsigned int> > >]’:
main-functions/pileup_readid.cpp:342:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
   86 |   unsigned int end;
      |                ^~~
main-functions/pileup_readid.cpp: In instantiation of ‘std::unordered_map<std::__cxx11::basic_string<char>, std::vector<std::vector<std::__cxx11::basic_string<char> > > > group_umis(const TableType&, int) [with TableType = std::unordered_map<std::__cxx11::basic_string<char>, std::unordered_map<std::__cxx11::basic_string<char>, std::array<unsigned int, 5> > >]’:
main-functions/pileup_readid.cpp:344:30:   required from here
main-functions/pileup_readid.cpp:86:16: warning: unused variable ‘end’ [-Wunused-variable]
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-junctions.cpp -o tests/test-junctions.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c tests/test-parsing.cpp -o tests/test-parsing.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/cigars.cpp -o utility/cigars.o
g++ -std=gnu++17 -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security  -c utility/edlib-1.2.7/edlib.cpp -o utility/edlib-1.2.7/edlib.o
gcc -std=gnu2x -I"/home/biocbuild/bbs-3.22-bioc/R/include" -DNDEBUG -pthread -D_FILE_OFFSET_BITS=64 -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rcpp/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/include' -I'/home/biocbuild/bbs-3.22-bioc/R/site-library/testthat/include' -I/usr/local/include    -fpic  -g -O2  -Wall -Werror=format-security -c utility/bam.c -o utility/bam.o
g++ -std=gnu++17 -shared -L/home/biocbuild/bbs-3.22-bioc/R/lib -L/usr/local/lib -o FLAMES.so RcppExports.o RcppFunctions.o classes/BamRecord.o classes/GFFRecord.o classes/GeneAnnotationParser.o classes/Isoforms.o classes/junctions.o main-functions/find_isoform.o main-functions/flexiplex.o main-functions/get_transcript_seq.o main-functions/group_bam2isoform.o main-functions/pileup_readid.o tests/test-junctions.o tests/test-parsing.o utility/cigars.o utility/edlib-1.2.7/edlib.o utility/bam.o -pthread -lz /home/biocbuild/bbs-3.22-bioc/R/site-library/Rhtslib/usrlib/libhts.a -lcurl -lbz2 -llzma -lz -L/home/biocbuild/bbs-3.22-bioc/R/lib -lR
if test -e "/usr/bin/strip" & test -e "/bin/uname" & [[ `uname` == "Linux" ]] ; then /usr/bin/strip --strip-debug FLAMES.so; fi
Building for x86_64
(cd submodule/minimap2 && make -f Makefile CFLAGS="-g -O2  -Wall -Werror=format-security -Wno-unused-result" minimap2)
make[1]: Entering directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  main.c -o main.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kthread.c -o kthread.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  kalloc.c -o kalloc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  misc.c -o misc.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  bseq.c -o bseq.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sketch.c -o sketch.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  sdust.c -o sdust.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  options.c -o options.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  index.c -o index.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  lchain.c -o lchain.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  align.c -o align.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  hit.c -o hit.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  seed.c -o seed.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  jump.c -o jump.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  map.c -o map.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  format.c -o format.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  pe.c -o pe.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  esterr.c -o esterr.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -DHAVE_KALLOC  splitidx.c -o splitidx.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -DHAVE_KALLOC  ksw2_ll_sse.c -o ksw2_ll_sse.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extz2_sse.c -o ksw2_extz2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_extd2_sse.c -o ksw2_extd2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_exts2_sse.c -o ksw2_exts2_sse41.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extz2_sse.c -o ksw2_extz2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_extd2_sse.c -o ksw2_extd2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse2 -mno-sse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH -DKSW_SSE2_ONLY  ksw2_exts2_sse.c -o ksw2_exts2_sse2.o
cc -c -g -O2  -Wall -Werror=format-security -Wno-unused-result -msse4.1 -DHAVE_KALLOC -DKSW_CPU_DISPATCH  ksw2_dispatch.c -o ksw2_dispatch.o
ar -csru libminimap2.a kthread.o kalloc.o misc.o bseq.o sketch.o sdust.o options.o index.o lchain.o align.o hit.o seed.o jump.o map.o format.o pe.o esterr.o splitidx.o ksw2_ll_sse.o ksw2_extz2_sse41.o ksw2_extd2_sse41.o ksw2_exts2_sse41.o ksw2_extz2_sse2.o ksw2_extd2_sse2.o ksw2_exts2_sse2.o ksw2_dispatch.o
ar: `u' modifier ignored since `D' is the default (see `U')
cc -g -O2  -Wall -Werror=format-security -Wno-unused-result main.o -o minimap2 -L. -lminimap2 -lm -lz -lpthread
make[1]: Leaving directory '/home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/submodule/minimap2'
echo "Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin"
Installing binary to /home/biocbuild/bbs-3.22-bioc/meat/FLAMES/src/../inst/bin
mkdir -p ../inst/bin
cp submodule/minimap2/minimap2 ../inst/bin/
Rust version too old for edition 2024, skipping oarfish installation.
installing to /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/libs
** R
** data
** inst
** byte-compile and prepare package for lazy loading
** help
*** installing help indices
*** copying figures
** building package indices
** installing vignettes
** testing if installed package can be loaded
* DONE (FLAMES)

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

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

R is a collaborative project with many contributors.
Type 'contributors()' for more information and
'citation()' on how to cite R or R packages in publications.

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af336b8324/config_file_690095.json 
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af336b8324/config_file_690095.json 
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af336b8324/config_file_690095.json 
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af9a0e201/config_file_690095.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/RtmptesnZ0/filea87af7a45ded2/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/RtmptesnZ0/filea87af4feb83e/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/RtmptesnZ0/filea87af4feb83e/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af2ea02eab/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af2ea02eab/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af2ea02eab/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af2ea02eab/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/RtmptesnZ0/filea87af26c2472d/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/RtmptesnZ0/filea87af788e89cb/config_file_690095.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 @ Sun Nov 16 23:58:55 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmptesnZ0/filea87af788e89cb/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmptesnZ0/filea87af788e89cb/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmptesnZ0/filea87af788e89cb/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Sun Nov 16 23:58:56 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[23:59:03] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:59:03] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:59:03] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:59:03] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:59:05] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.

[23:59:05] WARNING: src/learner.cc:553: 
  If you are loading a serialized model (like pickle in Python, RDS in R) generated by
  older XGBoost, please export the model by calling `Booster.save_model` from that version
  first, then load it back in current version. See:

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

  for more details about differences between saving model and serializing.


  |                                                                            
  |=======================                                               |  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 @ Sun Nov 16 23:59:21 2025 -------------------
Realigning sample sample1 -> /tmp/RtmptesnZ0/filea87af788e89cb/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmptesnZ0/filea87af788e89cb/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmptesnZ0/filea87af788e89cb/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Sun Nov 16 23:59:21 2025 ----------
2025-11-17T04:59:21.624623Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:59:21.625096Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af788e89cb/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:59:21.625110Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:59:21.625119Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:59:21.625180Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:59:21.625187Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:59:21.626676Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:59:21.626815Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:59:21.626843Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-11-17T04:59:21.626859Z  INFO oarfish::bulk: number of aligned reads : 96
2025-11-17T04:59:21.626862Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-17T04:59:21.627473Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:59:21.634973Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:59:21.635426Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af788e89cb/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:59:21.635438Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:59:21.635442Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:59:21.635505Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:59:21.635510Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:59:21.637074Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T04:59:21.637224Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-11-17T04:59:21.637251Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-11-17T04:59:21.637253Z  INFO oarfish::bulk: number of aligned reads : 95
2025-11-17T04:59:21.637255Z  INFO oarfish::bulk: number of unique alignments : 82
2025-11-17T04:59:21.637864Z  INFO oarfish: oarfish completed successfully.
2025-11-17T04:59:21.645361Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T04:59:21.645758Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af788e89cb/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T04:59:21.645767Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T04:59:21.645771Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T04:59:21.645830Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T04:59:21.645836Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T04:59:21.648599Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-11-17T04:59:21.648781Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-11-17T04:59:21.648821Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-11-17T04:59:21.648823Z  INFO oarfish::bulk: number of aligned reads : 179
2025-11-17T04:59:21.648826Z  INFO oarfish::bulk: number of unique alignments : 143
2025-11-17T04:59:21.649534Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af70e2f8b8/config_file_690095.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 @ Sun Nov 16 23:59:21 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmptesnZ0/filea87af70e2f8b8/sample1_align2genome.bam
sample2 ->/tmp/RtmptesnZ0/filea87af70e2f8b8/sample2_align2genome.bam
sample3 ->/tmp/RtmptesnZ0/filea87af70e2f8b8/sample3_align2genome.bam
-- Running step: isoform_identification @ Sun Nov 16 23:59:41 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:00:01 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmptesnZ0/filea87af70e2f8b8/sample1_realign2transcript.bam
sample2 ->/tmp/RtmptesnZ0/filea87af70e2f8b8/sample2_realign2transcript.bam
sample3 ->/tmp/RtmptesnZ0/filea87af70e2f8b8/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:00:21 2025 ----------
2025-11-17T05:00:21.551387Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:00:21.551978Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af70e2f8b8/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:00:21.552002Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:00:21.552006Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:00:21.552073Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:00:21.552079Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:00:21.554019Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T05:00:21.554155Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 253   │
│ aligned fraction too low        │ 4     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 1     │
│ reads with valid best alignment │ 96    │
╰─────────────────────────────────┴───────╯

2025-11-17T05:00:21.554180Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-11-17T05:00:21.554183Z  INFO oarfish::bulk: number of aligned reads : 96
2025-11-17T05:00:21.554185Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-17T05:00:21.554757Z  INFO oarfish: oarfish completed successfully.
2025-11-17T05:00:21.562708Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:00:21.563060Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af70e2f8b8/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:00:21.563068Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:00:21.563071Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:00:21.563129Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:00:21.563138Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:00:21.564667Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T05:00:21.564802Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 251   │
│ aligned fraction too low        │ 5     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 2     │
│ reads with valid best alignment │ 95    │
╰─────────────────────────────────┴───────╯

2025-11-17T05:00:21.564832Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-11-17T05:00:21.564845Z  INFO oarfish::bulk: number of aligned reads : 95
2025-11-17T05:00:21.564848Z  INFO oarfish::bulk: number of unique alignments : 82
2025-11-17T05:00:21.565415Z  INFO oarfish: oarfish completed successfully.
2025-11-17T05:00:21.573147Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:00:21.573517Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af70e2f8b8/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:00:21.573527Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:00:21.573530Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:00:21.573592Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:00:21.573598Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:00:21.576330Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-11-17T05:00:21.576497Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 398   │
│ aligned fraction too low        │ 12    │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 5     │
│ reads with valid best alignment │ 179   │
╰─────────────────────────────────┴───────╯

2025-11-17T05:00:21.576525Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-11-17T05:00:21.576528Z  INFO oarfish::bulk: number of aligned reads : 179
2025-11-17T05:00:21.576530Z  INFO oarfish::bulk: number of unique alignments : 143
2025-11-17T05:00:21.577203Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af7aa27468/config_file_690095.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 @ Mon Nov 17 00:00:21 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmptesnZ0/filea87af7aa27468/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmptesnZ0/filea87af7aa27468/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmptesnZ0/filea87af7aa27468/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Nov 17 00:00:22 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:00:41 2025 -------------------
Realigning sample sample1 -> /tmp/RtmptesnZ0/filea87af7aa27468/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmptesnZ0/filea87af7aa27468/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmptesnZ0/filea87af7aa27468/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Nov 17 00:00:41 2025 ----------
00:00:41 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af60af5a2f/config_file_690095.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 @ Mon Nov 17 00:00:43 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmptesnZ0/filea87af60af5a2f/sample1_align2genome.bam
sample2 ->/tmp/RtmptesnZ0/filea87af60af5a2f/sample2_align2genome.bam
sample3 ->/tmp/RtmptesnZ0/filea87af60af5a2f/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Nov 17 00:01:01 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:01:19 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmptesnZ0/filea87af60af5a2f/sample1_realign2transcript.bam
sample2 ->/tmp/RtmptesnZ0/filea87af60af5a2f/sample2_realign2transcript.bam
sample3 ->/tmp/RtmptesnZ0/filea87af60af5a2f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:01:37 2025 ----------
00:01:37 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/RtmptesnZ0/filea87af7aa27468/sample1_realign2transcript.bam', '/tmp/RtmptesnZ0/filea87af7aa27468/sample2_realign2transcript.bam', '/tmp/RtmptesnZ0/filea87af7aa27468/sample3_realign2transcript.bam'] /tmp/RtmptesnZ0/filea87af7aa27468/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/RtmptesnZ0/filea87af12ffbce0/config_file_690095.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 @ Mon Nov 17 00:01:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmptesnZ0/filea87af12ffbce0/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmptesnZ0/filea87af12ffbce0/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmptesnZ0/filea87af12ffbce0/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Nov 17 00:01:39 2025 -------------
Inputs:  ['/tmp/RtmptesnZ0/filea87af60af5a2f/sample1_realign2transcript.bam', '/tmp/RtmptesnZ0/filea87af60af5a2f/sample2_realign2transcript.bam', '/tmp/RtmptesnZ0/filea87af60af5a2f/sample3_realign2transcript.bam'] /tmp/RtmptesnZ0/filea87af60af5a2f/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 @ Mon Nov 17 00:01:40 2025 -------------------
Realigning sample sample1 -> /tmp/RtmptesnZ0/filea87af12ffbce0/sample1_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/RtmptesnZ0/filea87af12ffbce0/sample2_realign2transcript.bam
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/RtmptesnZ0/filea87af12ffbce0/sample3_realign2transcript.bam
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Mon Nov 17 00:01:40 2025 ----------
2025-11-17T05:01:40.905561Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:01:40.905965Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af12ffbce0/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T05:01:40.905986Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:01:40.905991Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:01:40.906067Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:01:40.906075Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T05:01:40.908665Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T05:01:40.908811Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-11-17T05:01:40.908833Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-11-17T05:01:40.908836Z  INFO oarfish::bulk: number of aligned reads : 98
2025-11-17T05:01:40.908838Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-17T05:01:40.909486Z  INFO oarfish: oarfish completed successfully.
2025-11-17T05:01:40.918214Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:01:40.918716Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af12ffbce0/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T05:01:40.918725Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:01:40.918729Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:01:40.918809Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:01:40.918816Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T05:01:40.921438Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T05:01:40.921574Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-11-17T05:01:40.921601Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-11-17T05:01:40.921603Z  INFO oarfish::bulk: number of aligned reads : 97
2025-11-17T05:01:40.921605Z  INFO oarfish::bulk: number of unique alignments : 79
2025-11-17T05:01:40.922215Z  INFO oarfish: oarfish completed successfully.
2025-11-17T05:01:40.930105Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:01:40.930472Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af12ffbce0/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T05:01:40.930491Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:01:40.930494Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:01:40.930574Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:01:40.930580Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T05:01:40.934856Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T05:01:40.935014Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-11-17T05:01:40.935042Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-11-17T05:01:40.935045Z  INFO oarfish::bulk: number of aligned reads : 187
2025-11-17T05:01:40.935047Z  INFO oarfish::bulk: number of unique alignments : 140
2025-11-17T05:01:40.935721Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af72a5db9e/config_file_690095.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 @ Mon Nov 17 00:01:41 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmptesnZ0/filea87af72a5db9e/sample1_align2genome.bam
sample2 ->/tmp/RtmptesnZ0/filea87af72a5db9e/sample2_align2genome.bam
sample3 ->/tmp/RtmptesnZ0/filea87af72a5db9e/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Nov 17 00:01:59 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:02:00 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmptesnZ0/filea87af72a5db9e/sample1_realign2transcript.bam
sample2 ->/tmp/RtmptesnZ0/filea87af72a5db9e/sample2_realign2transcript.bam
sample3 ->/tmp/RtmptesnZ0/filea87af72a5db9e/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:02:19 2025 ----------
2025-11-17T05:02:19.788449Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:19.788989Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af72a5db9e/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T05:02:19.789001Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:19.789015Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:19.789099Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:19.789106Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T05:02:19.791674Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T05:02:19.791808Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 707   │
│ aligned fraction too low        │ 2     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 98    │
╰─────────────────────────────────┴───────╯

2025-11-17T05:02:19.791830Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-11-17T05:02:19.791832Z  INFO oarfish::bulk: number of aligned reads : 98
2025-11-17T05:02:19.791835Z  INFO oarfish::bulk: number of unique alignments : 86
2025-11-17T05:02:19.792471Z  INFO oarfish: oarfish completed successfully.
2025-11-17T05:02:19.802162Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:19.802604Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af72a5db9e/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T05:02:19.802613Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:19.802616Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:19.802693Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:19.802700Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T05:02:19.805394Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T05:02:19.805528Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 701   │
│ aligned fraction too low        │ 3     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 97    │
╰─────────────────────────────────┴───────╯

2025-11-17T05:02:19.805553Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-11-17T05:02:19.805555Z  INFO oarfish::bulk: number of aligned reads : 97
2025-11-17T05:02:19.805568Z  INFO oarfish::bulk: number of unique alignments : 79
2025-11-17T05:02:19.806242Z  INFO oarfish: oarfish completed successfully.
2025-11-17T05:02:19.815488Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:02:19.815856Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af72a5db9e/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-11-17T05:02:19.815863Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:02:19.815866Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:02:19.815941Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:02:19.815947Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T05:02:19.820308Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-11-17T05:02:19.820484Z  INFO oarfish::bulk: 
discard_table: 
╭─────────────────────────────────┬───────╮
│ reason                          │ count │
├─────────────────────────────────┼───────┤
│ too far from 5' end             │ 0     │
│ too far from 3' end             │ 0     │
│ score too low                   │ 1060  │
│ aligned fraction too low        │ 6     │
│ aligned length too short        │ 0     │
│ inconsistent orientation        │ 0     │
│ supplementary alignment         │ 0     │
│ reads with valid best alignment │ 187   │
╰─────────────────────────────────┴───────╯

2025-11-17T05:02:19.820512Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-11-17T05:02:19.820515Z  INFO oarfish::bulk: number of aligned reads : 187
2025-11-17T05:02:19.820518Z  INFO oarfish::bulk: number of unique alignments : 140
2025-11-17T05:02:19.821253Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af7a8f5f10/config_file_690095.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 @ Mon Nov 17 00:02:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/RtmptesnZ0/filea87af7a8f5f10/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/RtmptesnZ0/filea87af7a8f5f10/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/RtmptesnZ0/filea87af7a8f5f10/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Nov 17 00:02:20 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:02:21 2025 -------------------
Realigning sample sample1 -> /tmp/RtmptesnZ0/filea87af7a8f5f10/sample1_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/RtmptesnZ0/filea87af7a8f5f10/sample2_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/RtmptesnZ0/filea87af7a8f5f10/sample3_realign2transcript.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Mon Nov 17 00:02:21 2025 ----------
00:02:21 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af3242c15f/config_file_690095.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 @ Mon Nov 17 00:02:22 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/RtmptesnZ0/filea87af3242c15f/sample1_align2genome.bam
sample2 ->/tmp/RtmptesnZ0/filea87af3242c15f/sample2_align2genome.bam
sample3 ->/tmp/RtmptesnZ0/filea87af3242c15f/sample3_align2genome.bam
-- Running step: isoform_identification @ Mon Nov 17 00:02:42 2025 -------------
Inputs:  ['/tmp/RtmptesnZ0/filea87af7a8f5f10/sample1_realign2transcript.bam', '/tmp/RtmptesnZ0/filea87af7a8f5f10/sample2_realign2transcript.bam', '/tmp/RtmptesnZ0/filea87af7a8f5f10/sample3_realign2transcript.bam'] /tmp/RtmptesnZ0/filea87af7a8f5f10/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 @ Mon Nov 17 00:02:42 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/RtmptesnZ0/filea87af3242c15f/sample1_realign2transcript.bam
sample2 ->/tmp/RtmptesnZ0/filea87af3242c15f/sample2_realign2transcript.bam
sample3 ->/tmp/RtmptesnZ0/filea87af3242c15f/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:03:01 2025 ----------
00:03:01 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af43e0fd/config_file_690095.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 @ Mon Nov 17 00:03:02 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af43e0fd/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 @ Mon Nov 17 00:03:02 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmptesnZ0/filea87af43e0fd/matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af43e0fd/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Nov 17 00:03:02 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:03:11 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af43e0fd/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af43e0fd/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmptesnZ0/filea87af43e0fd/matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af43e0fd/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 @ Mon Nov 17 00:03:12 2025 ----------
2025-11-17T05:03:12.134319Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:03:12.134826Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af43e0fd/realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:03:12.134834Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:03:12.134837Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:03:12.134893Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:03:12.134899Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:03:12.142080Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af2177d22a/config_file_690095.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 @ Mon Nov 17 00:03:12 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af2177d22a/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 @ Mon Nov 17 00:03:12 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af2177d22a/matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af2177d22a/align2genome.bam
-- Running step: isoform_identification @ Mon Nov 17 00:03:30 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:03:39 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af2177d22a/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af2177d22a/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af2177d22a/matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af2177d22a/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:03:59 2025 ----------
2025-11-17T05:03:59.147165Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:03:59.147588Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af2177d22a/realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:03:59.147598Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:03:59.147602Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:03:59.147661Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:03:59.147668Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:03:59.154091Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af52a021ef/config_file_690095.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 @ Mon Nov 17 00:03:59 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af52a021ef/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 @ Mon Nov 17 00:03:59 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmptesnZ0/filea87af52a021ef/matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af52a021ef/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 @ Mon Nov 17 00:04:00 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:04:09 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af52a021ef/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af52a021ef/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmptesnZ0/filea87af52a021ef/matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af52a021ef/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 @ Mon Nov 17 00:04:10 2025 ----------
00:04:10 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Inputs:  ['/tmp/RtmptesnZ0/filea87af3242c15f/sample1_realign2transcript.bam', '/tmp/RtmptesnZ0/filea87af3242c15f/sample2_realign2transcript.bam', '/tmp/RtmptesnZ0/filea87af3242c15f/sample3_realign2transcript.bam'] /tmp/RtmptesnZ0/filea87af3242c15f/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/RtmptesnZ0/filea87af3a1bc762/config_file_690095.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 @ Mon Nov 17 00:04:10 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af3a1bc762/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 @ Mon Nov 17 00:04:11 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af3a1bc762/matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af3a1bc762/align2genome.bam
-- Running step: isoform_identification @ Mon Nov 17 00:04:28 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---
Detected 2 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:04:39 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af3a1bc762/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af3a1bc762/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af3a1bc762/matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af3a1bc762/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:04:56 2025 ----------
00:04:56 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af32801519/config_file_690095.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 @ Mon Nov 17 00:04:57 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af32801519/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 @ Mon Nov 17 00:04:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmptesnZ0/filea87af32801519/matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af32801519/align2genome.bam
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: isoform_identification @ Mon Nov 17 00:04:57 2025 -------------
	Counter({'counted_reads': 354, 'not_enough_coverage': 12, 'unmapped': 6})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:04:58 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af32801519/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af32801519/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmptesnZ0/filea87af32801519/matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af32801519/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 @ Mon Nov 17 00:04:58 2025 ----------
2025-11-17T05:04:58.596914Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:04:58.597553Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af32801519/realign2transcript.bam, contains 10 reference sequences.
2025-11-17T05:04:58.597565Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:04:58.597570Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:04:58.597648Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:04:58.597656Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T05:04:58.608521Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af1cce7ac3/config_file_690095.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 @ Mon Nov 17 00:04:59 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af1cce7ac3/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 @ Mon Nov 17 00:04:59 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af1cce7ac3/matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af1cce7ac3/align2genome.bam
-- Running step: isoform_identification @ Mon Nov 17 00:05:16 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:05:17 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af1cce7ac3/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af1cce7ac3/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af1cce7ac3/matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af1cce7ac3/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:05:34 2025 ----------
2025-11-17T05:05:34.698158Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:05:34.698583Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af1cce7ac3/realign2transcript.bam, contains 10 reference sequences.
2025-11-17T05:05:34.698593Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:05:34.698596Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:05:34.698669Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:05:34.698689Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-11-17T05:05:34.708288Z  INFO oarfish::single_cell: Processed 100 cells.
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af86c508f/config_file_690095.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 @ Mon Nov 17 00:05:35 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af86c508f/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 @ Mon Nov 17 00:05:35 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmptesnZ0/filea87af86c508f/matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af86c508f/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 @ Mon Nov 17 00:05:36 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:05:36 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af86c508f/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af86c508f/matched_reads_dedup.fastq.gz
	files not found
Realigning sample /tmp/RtmptesnZ0/filea87af86c508f/matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af86c508f/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 @ Mon Nov 17 00:05:36 2025 ----------
00:05:36 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af462d1042/config_file_690095.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 @ Mon Nov 17 00:05:37 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 8
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af462d1042/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 @ Mon Nov 17 00:05:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af462d1042/matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af462d1042/align2genome.bam
-- Running step: isoform_identification @ Mon Nov 17 00:05:55 2025 -------------
	Counter({'counted_reads': 368, 'unmapped': 4})
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:05:56 2025 -------------------
Checking for fastq file(s) /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/extdata/fastq/musc_rps24.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af462d1042/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af462d1042/matched_reads_dedup.fastq.gz
	files not found
Realignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af462d1042/matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af462d1042/realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:06:13 2025 ----------
00:06:13 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af34824696/config_file_690095.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 @ Mon Nov 17 00:06:14 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af34824696/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af34824696/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af34824696/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af34824696/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/RtmptesnZ0/filea87af34824696/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af34824696/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/RtmptesnZ0/filea87af34824696/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af34824696/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/RtmptesnZ0/filea87af34824696/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af34824696/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 @ Mon Nov 17 00:06:15 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmptesnZ0/filea87af34824696/sampleA_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af34824696/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmptesnZ0/filea87af34824696/sample1_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af34824696/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmptesnZ0/filea87af34824696/sample2_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af34824696/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmptesnZ0/filea87af34824696/sample3_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af34824696/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Nov 17 00:06:17 2025 ----------------
00:06:17 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmptesnZ0/filea87af34824696/sampleA_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af34824696/sample1_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af34824696/sample2_align2genome.bam', and
'/tmp/RtmptesnZ0/filea87af34824696/sample3_align2genome.bam'
	Counter({'counted_reads': 368, 'unmapped': 4})
parsing /tmp/RtmptesnZ0/filea87af34824696/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.07gene_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, 384178.21Read/s]
parsing /tmp/RtmptesnZ0/filea87af34824696/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.54gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1291031.77Read/s]
parsing /tmp/RtmptesnZ0/filea87af34824696/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.93gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1242535.85Read/s]
parsing /tmp/RtmptesnZ0/filea87af34824696/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 711912.55Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:06:19 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:06:43 2025 -------------------
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af34824696/fastq, /tmp/RtmptesnZ0/filea87af34824696/fastq/sample1.fq.gz, /tmp/RtmptesnZ0/filea87af34824696/fastq/sample2.fq.gz, /tmp/RtmptesnZ0/filea87af34824696/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af34824696/sampleA_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af34824696/sample1_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af34824696/sample2_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af34824696/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af34824696/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af34824696/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af34824696/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af34824696/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmptesnZ0/filea87af34824696/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af34824696/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmptesnZ0/filea87af34824696/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af34824696/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmptesnZ0/filea87af34824696/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af34824696/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmptesnZ0/filea87af34824696/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af34824696/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Nov 17 00:06:43 2025 ----------
2025-11-17T05:06:43.925875Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:06:43.926383Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af34824696/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:06:43.926397Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:06:43.926415Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:06:43.926492Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:06:43.926498Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:06:43.932309Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-17T05:06:44.249302Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:06:44.249684Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af34824696/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:06:44.249693Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:06:44.249697Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:06:44.249765Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:06:44.249771Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:06:44.548152Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:06:44.548526Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af34824696/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:06:44.548534Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:06:44.548537Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:06:44.548599Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:06:44.548605Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:06:44.853061Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:06:44.853446Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af34824696/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:06:44.853457Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:06:44.853461Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:06:44.853514Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:06:44.853520Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af56dec907/config_file_690095.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 @ Mon Nov 17 00:06:45 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af56dec907/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af56dec907/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af56dec907/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af56dec907/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/RtmptesnZ0/filea87af56dec907/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af56dec907/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/RtmptesnZ0/filea87af56dec907/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af56dec907/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/RtmptesnZ0/filea87af56dec907/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af56dec907/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 @ Mon Nov 17 00:06:46 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af56dec907/sampleA_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af56dec907/sampleA_align2genome.bam
/tmp/RtmptesnZ0/filea87af56dec907/sample1_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af56dec907/sample1_align2genome.bam
/tmp/RtmptesnZ0/filea87af56dec907/sample2_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af56dec907/sample2_align2genome.bam
/tmp/RtmptesnZ0/filea87af56dec907/sample3_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af56dec907/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Nov 17 00:07:06 2025 ----------------
00:07:06 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmptesnZ0/filea87af56dec907/sampleA_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af56dec907/sample1_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af56dec907/sample2_align2genome.bam', and
'/tmp/RtmptesnZ0/filea87af56dec907/sample3_align2genome.bam'
parsing /tmp/RtmptesnZ0/filea87af56dec907/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.47gene_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, 384840.90Read/s]
parsing /tmp/RtmptesnZ0/filea87af56dec907/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.02gene_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, 1416037.81Read/s]
parsing /tmp/RtmptesnZ0/filea87af56dec907/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 46.96gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1269616.18Read/s]
parsing /tmp/RtmptesnZ0/filea87af56dec907/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 32.64gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 672207.19Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:07:07 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:07:32 2025 -------------------
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af56dec907/fastq, /tmp/RtmptesnZ0/filea87af56dec907/fastq/sample1.fq.gz, /tmp/RtmptesnZ0/filea87af56dec907/fastq/sample2.fq.gz, /tmp/RtmptesnZ0/filea87af56dec907/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af56dec907/sampleA_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af56dec907/sample1_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af56dec907/sample2_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af56dec907/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af56dec907/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af56dec907/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af56dec907/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af56dec907/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af56dec907/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af56dec907/sampleA_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af56dec907/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af56dec907/sample1_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af56dec907/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af56dec907/sample2_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af56dec907/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af56dec907/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:07:50 2025 ----------
2025-11-17T05:07:50.781770Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:07:50.782246Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af56dec907/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:07:50.782258Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:07:50.782262Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:07:50.782326Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:07:50.782332Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:07:50.788545Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-17T05:07:51.142071Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:07:51.142535Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af56dec907/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:07:51.142547Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:07:51.142551Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:07:51.142613Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:07:51.142618Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:07:51.484697Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:07:51.485096Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af56dec907/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:07:51.485104Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:07:51.485107Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:07:51.485176Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:07:51.485185Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-11-17T05:07:51.854426Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:07:51.854881Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af56dec907/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-11-17T05:07:51.854890Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:07:51.854893Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:07:51.854957Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:07:51.854962Z  INFO oarfish: parsed reference information for 5 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af7fe893dd/config_file_690095.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 @ Mon Nov 17 00:07:52 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af7fe893dd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af7fe893dd/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af7fe893dd/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af7fe893dd/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 @ Mon Nov 17 00:07:53 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/sample1_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/sample2_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/sample3_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af7fe893dd/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 @ Mon Nov 17 00:07:54 2025 ----------------
00:07:54 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af7fe893dd/sample1_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af7fe893dd/sample2_align2genome.bam', and
'/tmp/RtmptesnZ0/filea87af7fe893dd/sample3_align2genome.bam'
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.81gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 368102.23Read/s]
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.36gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1193869.98Read/s]
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.55gene_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, 1097870.38Read/s]
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.65gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 650885.16Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:07:55 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:08:18 2025 -------------------
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af7fe893dd/fastq, /tmp/RtmptesnZ0/filea87af7fe893dd/fastq/sample1.fq.gz, /tmp/RtmptesnZ0/filea87af7fe893dd/fastq/sample2.fq.gz, /tmp/RtmptesnZ0/filea87af7fe893dd/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af7fe893dd/sample1_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af7fe893dd/sample2_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af7fe893dd/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af7fe893dd/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af7fe893dd/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af7fe893dd/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af7fe893dd/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/RtmptesnZ0/filea87af7fe893dd/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af7fe893dd/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 @ Mon Nov 17 00:08:19 2025 ----------
00:08:19 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af7fe893dd/sampleA_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample1_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af7fe893dd/sample1_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample2_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af7fe893dd/sample2_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample3_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af7fe893dd/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af7fe893dd/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af3e58d351/config_file_690095.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 @ Mon Nov 17 00:08:21 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af3e58d351/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af3e58d351/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af3e58d351/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af3e58d351/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/RtmptesnZ0/filea87af3e58d351/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af3e58d351/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/RtmptesnZ0/filea87af3e58d351/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af3e58d351/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/RtmptesnZ0/filea87af3e58d351/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af3e58d351/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 @ Mon Nov 17 00:08:22 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af3e58d351/sampleA_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af3e58d351/sampleA_align2genome.bam
/tmp/RtmptesnZ0/filea87af3e58d351/sample1_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af3e58d351/sample1_align2genome.bam
/tmp/RtmptesnZ0/filea87af3e58d351/sample2_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af3e58d351/sample2_align2genome.bam
/tmp/RtmptesnZ0/filea87af3e58d351/sample3_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af3e58d351/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Nov 17 00:08:41 2025 ----------------
00:08:41 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmptesnZ0/filea87af3e58d351/sampleA_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af3e58d351/sample1_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af3e58d351/sample2_align2genome.bam', and
'/tmp/RtmptesnZ0/filea87af3e58d351/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/RtmptesnZ0/filea87af3e58d351/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.39gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 332448.56Read/s]
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.52gene_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, 1275639.90Read/s]
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.81gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1217575.48Read/s]
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.70gene_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, 696496.84Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:08:42 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
-- Running step: read_realignment @ Mon Nov 17 00:09:04 2025 -------------------
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af3e58d351/fastq, /tmp/RtmptesnZ0/filea87af3e58d351/fastq/sample1.fq.gz, /tmp/RtmptesnZ0/filea87af3e58d351/fastq/sample2.fq.gz, /tmp/RtmptesnZ0/filea87af3e58d351/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af3e58d351/sampleA_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af3e58d351/sample1_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af3e58d351/sample2_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af3e58d351/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af3e58d351/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af3e58d351/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af3e58d351/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af3e58d351/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af3e58d351/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af3e58d351/sampleA_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af3e58d351/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af3e58d351/sample1_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af3e58d351/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af3e58d351/sample2_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af3e58d351/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af3e58d351/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:09:22 2025 ----------
00:09:22 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sampleA_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af3e58d351/sampleA_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample1_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af3e58d351/sample1_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample2_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af3e58d351/sample2_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample3_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af3e58d351/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af3e58d351/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af31daec03/config_file_690095.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 @ Mon Nov 17 00:09:24 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af31daec03/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af31daec03/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af31daec03/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af31daec03/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/RtmptesnZ0/filea87af31daec03/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af31daec03/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/RtmptesnZ0/filea87af31daec03/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af31daec03/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/RtmptesnZ0/filea87af31daec03/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af31daec03/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 @ Mon Nov 17 00:09:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmptesnZ0/filea87af31daec03/sampleA_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af31daec03/sampleA_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmptesnZ0/filea87af31daec03/sample1_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af31daec03/sample1_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmptesnZ0/filea87af31daec03/sample2_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af31daec03/sample2_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/RtmptesnZ0/filea87af31daec03/sample3_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af31daec03/sample3_align2genome.bam
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Mon Nov 17 00:09:26 2025 ----------------
00:09:26 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmptesnZ0/filea87af31daec03/sampleA_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af31daec03/sample1_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af31daec03/sample2_align2genome.bam', and
'/tmp/RtmptesnZ0/filea87af31daec03/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/RtmptesnZ0/filea87af31daec03/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.67gene_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, 416283.30Read/s]
parsing /tmp/RtmptesnZ0/filea87af31daec03/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.35gene_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, 1431698.53Read/s]
parsing /tmp/RtmptesnZ0/filea87af31daec03/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.03gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1297260.92Read/s]
parsing /tmp/RtmptesnZ0/filea87af31daec03/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.08gene_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, 753883.10Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:09:27 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:09:28 2025 -------------------
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af31daec03/fastq, /tmp/RtmptesnZ0/filea87af31daec03/fastq/sample1.fq.gz, /tmp/RtmptesnZ0/filea87af31daec03/fastq/sample2.fq.gz, /tmp/RtmptesnZ0/filea87af31daec03/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af31daec03/sampleA_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af31daec03/sample1_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af31daec03/sample2_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af31daec03/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af31daec03/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af31daec03/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af31daec03/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af31daec03/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmptesnZ0/filea87af31daec03/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af31daec03/sampleA_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmptesnZ0/filea87af31daec03/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af31daec03/sample1_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmptesnZ0/filea87af31daec03/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af31daec03/sample2_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/RtmptesnZ0/filea87af31daec03/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af31daec03/sample3_realign2transcript.bam
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Mon Nov 17 00:09:29 2025 ----------
2025-11-17T05:09:30.009285Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:09:30.009692Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af31daec03/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:09:30.009702Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:09:30.009717Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:09:30.009805Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:09:30.009813Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:09:30.021286Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-17T05:09:30.584328Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:09:30.584736Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af31daec03/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:09:30.584744Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:09:30.584746Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:09:30.584824Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:09:30.584831Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:09:31.094333Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:09:31.094793Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af31daec03/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:09:31.094802Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:09:31.094810Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:09:31.094890Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:09:31.094898Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:09:31.647054Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:09:31.647480Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af31daec03/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:09:31.647491Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:09:31.647495Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:09:31.647586Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:09:31.647594Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af59c94f30/config_file_690095.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 @ Mon Nov 17 00:09:32 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af59c94f30/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af59c94f30/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af59c94f30/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af59c94f30/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/RtmptesnZ0/filea87af59c94f30/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af59c94f30/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/RtmptesnZ0/filea87af59c94f30/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af59c94f30/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/RtmptesnZ0/filea87af59c94f30/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af59c94f30/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 @ Mon Nov 17 00:09:33 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af59c94f30/sampleA_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af59c94f30/sampleA_align2genome.bam
/tmp/RtmptesnZ0/filea87af59c94f30/sample1_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af59c94f30/sample1_align2genome.bam
/tmp/RtmptesnZ0/filea87af59c94f30/sample2_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af59c94f30/sample2_align2genome.bam
/tmp/RtmptesnZ0/filea87af59c94f30/sample3_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af59c94f30/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Nov 17 00:09:52 2025 ----------------
00:09:52 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmptesnZ0/filea87af59c94f30/sampleA_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af59c94f30/sample1_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af59c94f30/sample2_align2genome.bam', and
'/tmp/RtmptesnZ0/filea87af59c94f30/sample3_align2genome.bam'
parsing /tmp/RtmptesnZ0/filea87af59c94f30/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 16.12gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:705: RuntimeWarning: More than 20 figures have been opened. Figures created through the pyplot interface (`matplotlib.pyplot.figure`) are retained until explicitly closed and may consume too much memory. (To control this warning, see the rcParam `figure.max_open_warning`). Consider using `matplotlib.pyplot.close()`.
  plt.figure(figsize=(8, 6))
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 382510.49Read/s]
parsing /tmp/RtmptesnZ0/filea87af59c94f30/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.38gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1318300.23Read/s]
parsing /tmp/RtmptesnZ0/filea87af59c94f30/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.79gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1227265.92Read/s]
parsing /tmp/RtmptesnZ0/filea87af59c94f30/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.24gene_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, 770105.76Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:09:53 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:09:53 2025 -------------------
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af59c94f30/fastq, /tmp/RtmptesnZ0/filea87af59c94f30/fastq/sample1.fq.gz, /tmp/RtmptesnZ0/filea87af59c94f30/fastq/sample2.fq.gz, /tmp/RtmptesnZ0/filea87af59c94f30/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af59c94f30/sampleA_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af59c94f30/sample1_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af59c94f30/sample2_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af59c94f30/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af59c94f30/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af59c94f30/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af59c94f30/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af59c94f30/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af59c94f30/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af59c94f30/sampleA_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af59c94f30/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af59c94f30/sample1_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af59c94f30/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af59c94f30/sample2_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af59c94f30/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af59c94f30/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:10:14 2025 ----------
2025-11-17T05:10:14.564088Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:10:14.564693Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af59c94f30/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:10:14.564706Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:10:14.564710Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:10:14.564805Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:10:14.564813Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:10:14.576789Z  INFO oarfish::single_cell: Processed 100 cells.
2025-11-17T05:10:15.208328Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:10:15.208704Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af59c94f30/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:10:15.208712Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:10:15.208715Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:10:15.208791Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:10:15.208799Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:10:15.829997Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:10:15.830476Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af59c94f30/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:10:15.830489Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:10:15.830492Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:10:15.830573Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:10:15.830581Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-11-17T05:10:16.416603Z  INFO oarfish: setting user-provided filter parameters.
2025-11-17T05:10:16.417225Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/RtmptesnZ0/filea87af59c94f30/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-11-17T05:10:16.417237Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-11-17T05:10:16.417240Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-11-17T05:10:16.417318Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-11-17T05:10:16.417326Z  INFO oarfish: parsed reference information for 14 transcripts.
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af76a0fd2d/config_file_690095.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 @ Mon Nov 17 00:10:17 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af76a0fd2d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af76a0fd2d/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af76a0fd2d/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af76a0fd2d/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 @ Mon Nov 17 00:10:17 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/sample1_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/sample2_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/sample3_matched_reads.fastq.gz -> /tmp/RtmptesnZ0/filea87af76a0fd2d/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 @ Mon Nov 17 00:10:19 2025 ----------------
00:10:19 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af76a0fd2d/sample1_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af76a0fd2d/sample2_align2genome.bam', and
'/tmp/RtmptesnZ0/filea87af76a0fd2d/sample3_align2genome.bam'
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.75gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 437234.59Read/s]
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  8.74gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  8.72gene_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, 1460208.88Read/s]
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.43gene_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, 1388106.96Read/s]
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.09gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 780713.28Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:10:20 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:10:20 2025 -------------------
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af76a0fd2d/fastq, /tmp/RtmptesnZ0/filea87af76a0fd2d/fastq/sample1.fq.gz, /tmp/RtmptesnZ0/filea87af76a0fd2d/fastq/sample2.fq.gz, /tmp/RtmptesnZ0/filea87af76a0fd2d/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af76a0fd2d/sample1_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af76a0fd2d/sample2_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af76a0fd2d/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af76a0fd2d/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af76a0fd2d/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af76a0fd2d/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/sample1_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/sample2_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af76a0fd2d/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/RtmptesnZ0/filea87af76a0fd2d/sample3_matched_reads_dedup.fastq.gz -> /tmp/RtmptesnZ0/filea87af76a0fd2d/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 @ Mon Nov 17 00:10:21 2025 ----------
00:10:21 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af76a0fd2d/sampleA_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample1_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af76a0fd2d/sample1_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample2_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af76a0fd2d/sample2_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample3_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af76a0fd2d/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af76a0fd2d/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/RtmptesnZ0/filea87af6873ee26/config_file_690095.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 @ Mon Nov 17 00:10:24 2025 ----------------
Using flexiplex for barcode demultiplexing.
FLEXIPLEX 0.96.2
Setting max barcode edit distance to 2
Setting max flanking sequence edit distance to 8
Setting read IDs to be  replaced
Setting number of threads to 1
Search pattern: 
primer: CTACACGACGCTCTTCCGATCT
BC: NNNNNNNNNNNNNNNN
UMI: NNNNNNNNNNNN
polyT: TTTTTTTTT
Setting known barcodes from /tmp/RtmptesnZ0/filea87af6873ee26/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af6873ee26/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af6873ee26/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/RtmptesnZ0/filea87af6873ee26/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/RtmptesnZ0/filea87af6873ee26/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af6873ee26/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/RtmptesnZ0/filea87af6873ee26/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af6873ee26/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/RtmptesnZ0/filea87af6873ee26/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/RtmptesnZ0/filea87af6873ee26/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 @ Mon Nov 17 00:10:25 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af6873ee26/sampleA_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af6873ee26/sampleA_align2genome.bam
/tmp/RtmptesnZ0/filea87af6873ee26/sample1_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af6873ee26/sample1_align2genome.bam
/tmp/RtmptesnZ0/filea87af6873ee26/sample2_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af6873ee26/sample2_align2genome.bam
/tmp/RtmptesnZ0/filea87af6873ee26/sample3_matched_reads.fastq.gz ->/tmp/RtmptesnZ0/filea87af6873ee26/sample3_align2genome.bam
-- Running step: gene_quantification @ Mon Nov 17 00:10:44 2025 ----------------
00:10:44 Mon Nov 17 2025 quantify genes 
Using BAM(s): '/tmp/RtmptesnZ0/filea87af6873ee26/sampleA_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af6873ee26/sample1_align2genome.bam',
'/tmp/RtmptesnZ0/filea87af6873ee26/sample2_align2genome.bam', and
'/tmp/RtmptesnZ0/filea87af6873ee26/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 20.68gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 396947.30Read/s]
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.48gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1401464.85Read/s]
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.91gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 1262128.07Read/s]
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.23gene_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, 747541.17Read/s]
-- Running step: isoform_identification @ Mon Nov 17 00:10:45 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
-- Running step: read_realignment @ Mon Nov 17 00:10:45 2025 -------------------
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af6873ee26/fastq, /tmp/RtmptesnZ0/filea87af6873ee26/fastq/sample1.fq.gz, /tmp/RtmptesnZ0/filea87af6873ee26/fastq/sample2.fq.gz, /tmp/RtmptesnZ0/filea87af6873ee26/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af6873ee26/sampleA_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af6873ee26/sample1_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af6873ee26/sample2_matched_reads.fastq.gz, /tmp/RtmptesnZ0/filea87af6873ee26/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/RtmptesnZ0/filea87af6873ee26/sampleA_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af6873ee26/sample1_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af6873ee26/sample2_matched_reads_dedup.fastq.gz, /tmp/RtmptesnZ0/filea87af6873ee26/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/RtmptesnZ0/filea87af6873ee26/sampleA_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af6873ee26/sampleA_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af6873ee26/sample1_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af6873ee26/sample1_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af6873ee26/sample2_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af6873ee26/sample2_realign2transcript.bam
/tmp/RtmptesnZ0/filea87af6873ee26/sample3_matched_reads_dedup.fastq.gz ->/tmp/RtmptesnZ0/filea87af6873ee26/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Mon Nov 17 00:11:05 2025 ----------
00:11:05 Mon Nov 17 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sampleA_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af6873ee26/sampleA_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample1_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af6873ee26/sample1_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample2_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af6873ee26/sample2_realign2transcript.bamdone
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample3_realign2transcript.bam...
parsing /tmp/RtmptesnZ0/filea87af6873ee26/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/RtmptesnZ0/filea87af6873ee26/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]

[ FAIL 0 | WARN 183 | SKIP 0 | PASS 59 ]
	Counter({'counted_reads': 358})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 176})
> 
> proc.time()
   user  system elapsed 
722.614  43.590 752.896