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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD check --install=check:FLAMES.install-out.txt --library=/home/biocbuild/bbs-3.22-bioc/R/site-library --timings FLAMES_2.3.4.tar.gz
###
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* using log directory ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck’
* using R version 4.5.1 (2025-06-13)
* 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 24.04.2 LTS
* using session charset: UTF-8
* checking for file ‘FLAMES/DESCRIPTION’ ... OK
* this is package ‘FLAMES’ version ‘2.3.4’
* package encoding: UTF-8
* checking package namespace information ... OK
* checking package dependencies ... INFO
Imports includes 50 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 ... OK
* 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  5.9Mb
  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’
MultiSampleSCPipeline: no visible global function definition for ‘new’
SingleCellPipeline: no visible global function definition for ‘new’
addRowRanges: no visible global function definition for ‘head’
addRowRanges: 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_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_barcode: no visible binding for global variable ‘Sample’
find_barcode: no visible binding for global variable ‘Outfile’
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 ‘head’
plot_spatial_pie: no visible binding for global variable ‘imageX’
plot_spatial_pie: no visible binding for global variable ‘imageY’
sc_mutations: no visible binding for global variable ‘mutation_index’
sc_mutations: no visible binding for global variable ‘bam_index’
sc_transcript_usage_chisq: no visible global function definition for
  ‘as’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_chisq: no visible binding for global variable
  ‘adj.p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘total’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘test’
sc_transcript_usage_permutation: no visible global function definition
  for ‘as’
sc_transcript_usage_permutation : <anonymous>: no visible global
  function definition for ‘as’
sc_transcript_usage_permutation : <anonymous> : <anonymous>: no visible
  global function definition for ‘na.omit’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘transcript’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘p.value’
sc_transcript_usage_permutation: no visible binding for global variable
  ‘adj.p.value’
variant_count_tb: no visible binding for global variable ‘barcode’
variant_count_tb: no visible binding for global variable ‘allele_count’
variant_count_tb: no visible binding for global variable
  ‘cell_total_reads’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘outdir’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.MultiSampleSCPipeline: no visible binding
  for global variable ‘barcodes_file’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘expect_cell_number’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘outdir’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘demultiplexed_fastq’
barcode_demultiplex,FLAMES.SingleCellPipeline: no visible binding for
  global variable ‘barcodes_file’
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’
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
  Outfile Reads Sample Type UMI UMI_count adj.p.value 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 everything expect_cell_number
  expr fastq filter_res genome_bam 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 single match
  reads starts_with 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")
  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
blaze                         5.323 20.531  14.850
plot_isoform_reduced_dim     23.643  0.590  24.234
find_variants                19.844  0.383  19.551
bulk_long_pipeline            2.798 16.546   3.010
sc_long_multisample_pipeline 12.211  5.009  11.756
MultiSampleSCPipeline        13.706  1.009  15.005
sc_DTU_analysis               8.617  1.603   8.548
create_sce_from_dir           6.159  3.027   6.939
plot_isoform_heatmap          7.225  0.178   7.404
SingleCellPipeline            4.731  1.436   4.257
sc_long_pipeline              4.319  1.383   3.872
* 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: 4 NOTEs
See
  ‘/home/biocbuild/bbs-3.22-bioc/meat/FLAMES.Rcheck/00check.log’
for details.

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###
### Running command:
###
###   /home/biocbuild/bbs-3.22-bioc/R/bin/R CMD INSTALL FLAMES
###
##############################################################################
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* installing to library ‘/home/biocbuild/bbs-3.22-bioc/R/site-library’
* installing *source* package ‘FLAMES’ ...
** this is package ‘FLAMES’ version ‘2.3.4’
** 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:122:21: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  122 |       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:163:22: warning: comparison of integer expressions of different signedness: ‘std::__cxx11::basic_string<char>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  163 |     if (seq.length() < umi_start + umi_length) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:187:36: warning: comparison of integer expressions of different signedness: ‘std::vector<int>::size_type’ {aka ‘long unsigned int’} and ‘int’ [-Wsign-compare]
  187 |     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:295:19: 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]
  295 |     if (i_pattern >= subpattern_ends[i_subpattern]) {
main-functions/flexiplex.cpp:354:22: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  354 |     if (editDistance == barcode.editd) {
      |         ~~~~~~~~~~~~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:356:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  356 |     } else if (editDistance < barcode.editd &&
      |                ~~~~~~~~~~~~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:357:29: warning: comparison of integer expressions of different signedness: ‘unsigned int’ and ‘int’ [-Wsign-compare]
  357 |                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:396:29: 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]
  396 |       if (barcode.flank_end == std::string::npos) {
      |           ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_stats(const std::string&, const std::vector<Barcode>&, std::ostream&)’:
main-functions/flexiplex.cpp:421:38: 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]
  421 |                << (barcode.flank_end == std::string::npos ? "True" : "False")
      |                    ~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp: In function ‘void print_read(const std::string&, const std::string&, const std::string&, const std::vector<Barcode>&, gzFile, std::unordered_set<std::__cxx11::basic_string<char> >&, bool, bool, bool)’:
main-functions/flexiplex.cpp:462:21: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  462 |   for (int b = 0; b < vec_bc.size(); b++) {
      |                   ~~^~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:477:32: 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]
  477 |     if (vec_bc.at(b).flank_end == std::string::npos) {
      |         ~~~~~~~~~~~~~~~~~~~~~~~^~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:482:23: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  482 |     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:740:25: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<std::vector<SearchResult> >::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  740 |       for (int t = 0; t < sr_v.size();
      |                       ~~^~~~~~~~~~~~~
main-functions/flexiplex.cpp:745:27: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<SearchResult>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  745 |         for (int r = 0; r < sr_v[t].size(); r++) { // loop over the reads
      |                         ~~^~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:747:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  747 |           for (int b = 0; b < sr_v[t][r].vec_bc_for.size(); b++)
      |                           ~~^~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
main-functions/flexiplex.cpp:749:29: warning: comparison of integer expressions of different signedness: ‘int’ and ‘std::vector<Barcode>::size_type’ {aka ‘long unsigned int’} [-Wsign-compare]
  749 |           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 (2025-06-13) -- "Great Square Root"
Copyright (C) 2025 The R Foundation for Statistical Computing
Platform: x86_64-pc-linux-gnu

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

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

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

> # This file is part of the standard setup for testthat.
> # It is recommended that you do not modify it.
> #
> # Where should you do additional test configuration?
> # Learn more about the roles of various files in:
> # * https://r-pkgs.org/tests.html
> # * https://testthat.r-lib.org/reference/test_package.html#special-files
> 
> library(testthat)
> library(FLAMES)
> 
> test_check("FLAMES")
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file79221f14ec32/config_file_496161.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/Rtmp8hsqBt/file792217ed74aea/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!
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/Rtmp8hsqBt/file7922133f82bdc/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!
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/Rtmp8hsqBt/file7922133f82bdc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922148b3677c/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922148b3677c/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922148b3677c/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922148b3677c/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!
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/Rtmp8hsqBt/file792215cbdc4d/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!
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/Rtmp8hsqBt/file792215cbdc4d/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922129713024/musc_rps24_1.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922129713024/musc_rps24_2.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922129713024/musc_rps24_3.fastq
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922129713024/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!
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/Rtmp8hsqBt/file792215cbdc4d/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!
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/Rtmp8hsqBt/file792214e57984f/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!
Skipping TSO trimming...
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file7922176e1a4e8/config_file_496161.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Aug 12 01:24:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample1_align2genome.bam
[M::mm_idx_gen::0.003*1.20] collected minimizers
[M::mm_idx_gen::0.003*1.16] sorted minimizers
[M::main::0.003*1.16] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.15] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.15] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922176e1a4e8/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922176e1a4e8/rps24.fa /tmp/Rtmp8hsqBt/file7922176e1a4e8/fastq/sample1.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.173 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample2_align2genome.bam
[M::mm_idx_gen::0.002*1.20] collected minimizers
[M::mm_idx_gen::0.002*1.15] sorted minimizers
[M::main::0.002*1.15] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.14] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.13] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.172*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922176e1a4e8/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922176e1a4e8/rps24.fa /tmp/Rtmp8hsqBt/file7922176e1a4e8/fastq/sample2.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.172 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample3_align2genome.bam
[M::mm_idx_gen::0.002*0.98] collected minimizers
[M::mm_idx_gen::0.003*0.99] sorted minimizers
[M::main::0.003*0.99] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*0.99] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*0.99] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.333*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922176e1a4e8/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922176e1a4e8/rps24.fa /tmp/Rtmp8hsqBt/file7922176e1a4e8/fastq/sample3.fq.gz
[M::main] Real time: 0.333 sec; CPU: 0.333 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Aug 12 01:24:44 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

  |                                                                            
  |                                                                      |   0%[01:24:52] 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.

[01:24:52] 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.

[01:24:52] 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.

[01:24:52] 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.

[01:24:54] 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.

[01:24:54] 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 ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:25:12 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample1_realign2transcript.bam
[M::mm_idx_gen::0.002*1.14] collected minimizers
[M::mm_idx_gen::0.002*1.13] sorted minimizers
[M::main::0.002*1.12] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.12] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.12] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.083*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922176e1a4e8/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922176e1a4e8/fastq/sample1.fq.gz
[M::main] Real time: 0.084 sec; CPU: 0.084 sec; Peak RSS: 0.003 GB
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*2.45] collected minimizers
[M::mm_idx_gen::0.001*2.13] sorted minimizers
[M::main::0.001*2.11] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*2.06] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*2.01] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.087*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922176e1a4e8/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922176e1a4e8/fastq/sample2.fq.gz
[M::main] Real time: 0.088 sec; CPU: 0.089 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.00] collected minimizers
[M::mm_idx_gen::0.001*1.00] sorted minimizers
[M::main::0.001*1.00] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.00] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.00] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.161*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922176e1a4e8/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922176e1a4e8/fastq/sample3.fq.gz
[M::main] Real time: 0.161 sec; CPU: 0.161 sec; Peak RSS: 0.005 GB
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Tue Aug 12 01:25:13 2025 ----------
2025-08-12T05:25:13.476137Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:25:13.476573Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:25:13.476582Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:25:13.476585Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:25:13.476651Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:25:13.476659Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:25:13.478120Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:25:13.478231Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-12T05:25:13.478249Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-08-12T05:25:13.478252Z  INFO oarfish::bulk: number of aligned reads : 96
2025-08-12T05:25:13.478254Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-12T05:25:13.478835Z  INFO oarfish: oarfish completed successfully.
2025-08-12T05:25:13.486427Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:25:13.486804Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:25:13.486816Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:25:13.486819Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:25:13.486888Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:25:13.486894Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:25:13.488453Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:25:13.488577Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-12T05:25:13.488597Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-08-12T05:25:13.488599Z  INFO oarfish::bulk: number of aligned reads : 95
2025-08-12T05:25:13.488602Z  INFO oarfish::bulk: number of unique alignments : 82
2025-08-12T05:25:13.489197Z  INFO oarfish: oarfish completed successfully.
2025-08-12T05:25:13.496453Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:25:13.496829Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922176e1a4e8/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:25:13.496840Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:25:13.496843Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:25:13.496904Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:25:13.496910Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:25:13.499556Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-08-12T05:25:13.499753Z  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   │
╰─────────────────────────────────┴───────╯

2025-08-12T05:25:13.499784Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-08-12T05:25:13.499786Z  INFO oarfish::bulk: number of aligned reads : 179
2025-08-12T05:25:13.499789Z  INFO oarfish::bulk: number of unique alignments : 143
2025-08-12T05:25:13.500460Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792212262b438/config_file_496161.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Aug 12 01:25:13 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp8hsqBt/file792212262b438/sample1_align2genome.bam
sample2 ->/tmp/Rtmp8hsqBt/file792212262b438/sample2_align2genome.bam
sample3 ->/tmp/Rtmp8hsqBt/file792212262b438/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Aug 12 01:25:37 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 ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:25:55 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp8hsqBt/file792212262b438/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp8hsqBt/file792212262b438/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp8hsqBt/file792212262b438/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:26:17 2025 ----------
2025-08-12T05:26:17.973451Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:26:17.973834Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file792212262b438/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:26:17.973845Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:26:17.973849Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:26:17.973903Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:26:17.973909Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:26:17.975502Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:26:17.975653Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-12T05:26:17.975692Z  INFO oarfish::bulk: Total number of alignment records : 116
2025-08-12T05:26:17.975695Z  INFO oarfish::bulk: number of aligned reads : 96
2025-08-12T05:26:17.975697Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-12T05:26:17.976326Z  INFO oarfish: oarfish completed successfully.
2025-08-12T05:26:17.985588Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:26:17.985962Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file792212262b438/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:26:17.985973Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:26:17.985976Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:26:17.986025Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:26:17.986030Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:26:17.987701Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:26:17.987828Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-12T05:26:17.987849Z  INFO oarfish::bulk: Total number of alignment records : 114
2025-08-12T05:26:17.987851Z  INFO oarfish::bulk: number of aligned reads : 95
2025-08-12T05:26:17.987853Z  INFO oarfish::bulk: number of unique alignments : 82
2025-08-12T05:26:17.988465Z  INFO oarfish: oarfish completed successfully.
2025-08-12T05:26:17.997206Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:26:17.997560Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file792212262b438/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:26:17.997568Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:26:17.997582Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:26:17.997643Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:26:17.997651Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:26:18.000415Z  INFO oarfish::alignment_parser: the alignment file contained 2 unmapped read records.
2025-08-12T05:26:18.000563Z  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   │
╰─────────────────────────────────┴───────╯

2025-08-12T05:26:18.000589Z  INFO oarfish::bulk: Total number of alignment records : 239
2025-08-12T05:26:18.000592Z  INFO oarfish::bulk: number of aligned reads : 179
2025-08-12T05:26:18.000594Z  INFO oarfish::bulk: number of unique alignments : 143
2025-08-12T05:26:18.001282Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792211e77403a/config_file_496161.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Aug 12 01:26:18 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp8hsqBt/file792211e77403a/sample1_align2genome.bam
[M::mm_idx_gen::0.001*0.92] collected minimizers
[M::mm_idx_gen::0.002*0.95] sorted minimizers
[M::main::0.002*0.95] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.96] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.96] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file792211e77403a/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file792211e77403a/rps24.fa /tmp/Rtmp8hsqBt/file792211e77403a/fastq/sample1.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.172 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp8hsqBt/file792211e77403a/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.61] collected minimizers
[M::mm_idx_gen::0.002*1.40] sorted minimizers
[M::main::0.002*1.40] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.37] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.35] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.170*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file792211e77403a/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file792211e77403a/rps24.fa /tmp/Rtmp8hsqBt/file792211e77403a/fastq/sample2.fq.gz
[M::main] Real time: 0.171 sec; CPU: 0.171 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp8hsqBt/file792211e77403a/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.40] collected minimizers
[M::mm_idx_gen::0.002*1.27] sorted minimizers
[M::main::0.002*1.27] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.25] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.23] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.333*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file792211e77403a/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file792211e77403a/rps24.fa /tmp/Rtmp8hsqBt/file792211e77403a/fastq/sample3.fq.gz
[M::main] Real time: 0.333 sec; CPU: 0.334 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Aug 12 01:26:19 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:26:36 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp8hsqBt/file792211e77403a/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*2.24] collected minimizers
[M::mm_idx_gen::0.002*1.82] sorted minimizers
[M::main::0.002*1.81] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.79] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.76] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.069*1.02] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file792211e77403a/transcript_assembly.fa /tmp/Rtmp8hsqBt/file792211e77403a/fastq/sample1.fq.gz
[M::main] Real time: 0.069 sec; CPU: 0.071 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmp8hsqBt/file792211e77403a/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.42] collected minimizers
[M::mm_idx_gen::0.001*1.33] sorted minimizers
[M::main::0.001*1.33] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.31] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.30] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.070*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file792211e77403a/transcript_assembly.fa /tmp/Rtmp8hsqBt/file792211e77403a/fastq/sample2.fq.gz
[M::main] Real time: 0.070 sec; CPU: 0.070 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmp8hsqBt/file792211e77403a/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.80] collected minimizers
[M::mm_idx_gen::0.001*1.60] sorted minimizers
[M::main::0.001*1.59] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.56] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.53] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.136*1.01] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file792211e77403a/transcript_assembly.fa /tmp/Rtmp8hsqBt/file792211e77403a/fastq/sample3.fq.gz
[M::main] Real time: 0.136 sec; CPU: 0.137 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Aug 12 01:26:37 2025 ----------
01:26:37 Tue Aug 12 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/Rtmp8hsqBt/file7922131a1bf9/config_file_496161.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Aug 12 01:26:38 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp8hsqBt/file7922131a1bf9/sample1_align2genome.bam
sample2 ->/tmp/Rtmp8hsqBt/file7922131a1bf9/sample2_align2genome.bam
sample3 ->/tmp/Rtmp8hsqBt/file7922131a1bf9/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Aug 12 01:27:03 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 4 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:27:20 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp8hsqBt/file7922131a1bf9/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp8hsqBt/file7922131a1bf9/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp8hsqBt/file7922131a1bf9/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:27:41 2025 ----------
01:27:41 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
Inputs:  ['/tmp/Rtmp8hsqBt/file792211e77403a/sample1_realign2transcript.bam', '/tmp/Rtmp8hsqBt/file792211e77403a/sample2_realign2transcript.bam', '/tmp/Rtmp8hsqBt/file792211e77403a/sample3_realign2transcript.bam'] /tmp/Rtmp8hsqBt/file792211e77403a/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/Rtmp8hsqBt/file79221fd0c05d/config_file_496161.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Aug 12 01:27:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp8hsqBt/file79221fd0c05d/sample1_align2genome.bam
[M::mm_idx_gen::0.001*1.84] collected minimizers
[M::mm_idx_gen::0.002*1.58] sorted minimizers
[M::main::0.002*1.57] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.54] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.51] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.170*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file79221fd0c05d/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file79221fd0c05d/rps24.fa /tmp/Rtmp8hsqBt/file79221fd0c05d/fastq/sample1.fq.gz
[M::main] Real time: 0.171 sec; CPU: 0.172 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp8hsqBt/file79221fd0c05d/sample2_align2genome.bam
[M::mm_idx_gen::0.001*1.01] collected minimizers
[M::mm_idx_gen::0.002*1.01] sorted minimizers
[M::main::0.002*1.01] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.01] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.01] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file79221fd0c05d/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file79221fd0c05d/rps24.fa /tmp/Rtmp8hsqBt/file79221fd0c05d/fastq/sample2.fq.gz
[M::main] Real time: 0.171 sec; CPU: 0.171 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp8hsqBt/file79221fd0c05d/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.55] collected minimizers
[M::mm_idx_gen::0.002*1.36] sorted minimizers
[M::main::0.002*1.36] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.33] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.31] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.332*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file79221fd0c05d/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file79221fd0c05d/rps24.fa /tmp/Rtmp8hsqBt/file79221fd0c05d/fastq/sample3.fq.gz
[M::main] Real time: 0.333 sec; CPU: 0.333 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Aug 12 01:27:43 2025 -------------
Inputs:  ['/tmp/Rtmp8hsqBt/file7922131a1bf9/sample1_realign2transcript.bam', '/tmp/Rtmp8hsqBt/file7922131a1bf9/sample2_realign2transcript.bam', '/tmp/Rtmp8hsqBt/file7922131a1bf9/sample3_realign2transcript.bam'] /tmp/Rtmp8hsqBt/file7922131a1bf9/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
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:27:44 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp8hsqBt/file79221fd0c05d/sample1_realign2transcript.bam
[M::mm_idx_gen::0.002*1.37] collected minimizers
[M::mm_idx_gen::0.002*1.30] sorted minimizers
[M::main::0.002*1.30] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.28] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.27] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.174*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file79221fd0c05d/transcript_assembly.fa /tmp/Rtmp8hsqBt/file79221fd0c05d/fastq/sample1.fq.gz
[M::main] Real time: 0.174 sec; CPU: 0.175 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample2 -> /tmp/Rtmp8hsqBt/file79221fd0c05d/sample2_realign2transcript.bam
[M::mm_idx_gen::0.002*1.16] collected minimizers
[M::mm_idx_gen::0.003*1.13] sorted minimizers
[M::main::0.003*1.13] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*1.13] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*1.13] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.177*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file79221fd0c05d/transcript_assembly.fa /tmp/Rtmp8hsqBt/file79221fd0c05d/fastq/sample2.fq.gz
[M::main] Real time: 0.177 sec; CPU: 0.178 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

Realigning sample sample3 -> /tmp/Rtmp8hsqBt/file79221fd0c05d/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.50] collected minimizers
[M::mm_idx_gen::0.002*1.40] sorted minimizers
[M::main::0.002*1.39] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.37] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.36] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.306*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file79221fd0c05d/transcript_assembly.fa /tmp/Rtmp8hsqBt/file79221fd0c05d/fastq/sample3.fq.gz
[M::main] Real time: 0.307 sec; CPU: 0.307 sec; Peak RSS: 0.004 GB
Skipped sorting BAM files.

-- Running step: transcript_quantification @ Tue Aug 12 01:27:44 2025 ----------
2025-08-12T05:27:44.849269Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:27:44.849763Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221fd0c05d/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-08-12T05:27:44.849775Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:27:44.849778Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:27:44.849856Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:27:44.849863Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-12T05:27:44.852529Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:27:44.852683Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-12T05:27:44.852712Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-08-12T05:27:44.852715Z  INFO oarfish::bulk: number of aligned reads : 98
2025-08-12T05:27:44.852718Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-12T05:27:44.853360Z  INFO oarfish: oarfish completed successfully.
2025-08-12T05:27:44.860988Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:27:44.861476Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221fd0c05d/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-08-12T05:27:44.861485Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:27:44.861488Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:27:44.861572Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:27:44.861579Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-12T05:27:44.864278Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:27:44.864398Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-12T05:27:44.864423Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-08-12T05:27:44.864426Z  INFO oarfish::bulk: number of aligned reads : 97
2025-08-12T05:27:44.864428Z  INFO oarfish::bulk: number of unique alignments : 79
2025-08-12T05:27:44.865026Z  INFO oarfish: oarfish completed successfully.
2025-08-12T05:27:44.872324Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:27:44.872892Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221fd0c05d/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-08-12T05:27:44.872904Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:27:44.872907Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:27:44.872973Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:27:44.872979Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-12T05:27:44.877463Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:27:44.877625Z  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   │
╰─────────────────────────────────┴───────╯

2025-08-12T05:27:44.877655Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-08-12T05:27:44.877657Z  INFO oarfish::bulk: number of aligned reads : 187
2025-08-12T05:27:44.877659Z  INFO oarfish::bulk: number of unique alignments : 140
2025-08-12T05:27:44.878360Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file79221316e5b0c/config_file_496161.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Aug 12 01:27:45 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp8hsqBt/file79221316e5b0c/sample1_align2genome.bam
sample2 ->/tmp/Rtmp8hsqBt/file79221316e5b0c/sample2_align2genome.bam
sample3 ->/tmp/Rtmp8hsqBt/file79221316e5b0c/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Aug 12 01:28:06 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:28:07 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp8hsqBt/file79221316e5b0c/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp8hsqBt/file79221316e5b0c/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp8hsqBt/file79221316e5b0c/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:28:31 2025 ----------
2025-08-12T05:28:31.407955Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:28:31.408433Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221316e5b0c/sample1_realign2transcript.bam, contains 10 reference sequences.
2025-08-12T05:28:31.408443Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:28:31.408447Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:28:31.408515Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:28:31.408522Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-12T05:28:31.411168Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:28:31.411298Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-12T05:28:31.411324Z  INFO oarfish::bulk: Total number of alignment records : 125
2025-08-12T05:28:31.411327Z  INFO oarfish::bulk: number of aligned reads : 98
2025-08-12T05:28:31.411330Z  INFO oarfish::bulk: number of unique alignments : 86
2025-08-12T05:28:31.411948Z  INFO oarfish: oarfish completed successfully.
2025-08-12T05:28:31.421937Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:28:31.422341Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221316e5b0c/sample2_realign2transcript.bam, contains 10 reference sequences.
2025-08-12T05:28:31.422349Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:28:31.422353Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:28:31.422424Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:28:31.422431Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-12T05:28:31.425127Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:28:31.425262Z  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    │
╰─────────────────────────────────┴───────╯

2025-08-12T05:28:31.425287Z  INFO oarfish::bulk: Total number of alignment records : 136
2025-08-12T05:28:31.425290Z  INFO oarfish::bulk: number of aligned reads : 97
2025-08-12T05:28:31.425292Z  INFO oarfish::bulk: number of unique alignments : 79
2025-08-12T05:28:31.425946Z  INFO oarfish: oarfish completed successfully.
2025-08-12T05:28:31.433857Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:28:31.434236Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221316e5b0c/sample3_realign2transcript.bam, contains 10 reference sequences.
2025-08-12T05:28:31.434244Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:28:31.434247Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:28:31.434310Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:28:31.434316Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-12T05:28:31.438588Z  INFO oarfish::alignment_parser: the alignment file contained 0 unmapped read records.
2025-08-12T05:28:31.438769Z  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   │
╰─────────────────────────────────┴───────╯

2025-08-12T05:28:31.438812Z  INFO oarfish::bulk: Total number of alignment records : 272
2025-08-12T05:28:31.438814Z  INFO oarfish::bulk: number of aligned reads : 187
2025-08-12T05:28:31.438816Z  INFO oarfish::bulk: number of unique alignments : 140
2025-08-12T05:28:31.439500Z  INFO oarfish: oarfish completed successfully.
Testing: run_FLAMES(BulkPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file7922155a1066/config_file_496161.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Aug 12 01:28:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample sample1 -> /tmp/Rtmp8hsqBt/file7922155a1066/sample1_align2genome.bam
[M::mm_idx_gen::0.002*1.33] collected minimizers
[M::mm_idx_gen::0.002*1.24] sorted minimizers
[M::main::0.002*1.24] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.22] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.21] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.171*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922155a1066/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922155a1066/rps24.fa /tmp/Rtmp8hsqBt/file7922155a1066/fastq/sample1.fq.gz
[M::main] Real time: 0.172 sec; CPU: 0.172 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample2 -> /tmp/Rtmp8hsqBt/file7922155a1066/sample2_align2genome.bam
[M::mm_idx_gen::0.003*1.25] collected minimizers
[M::mm_idx_gen::0.003*1.20] sorted minimizers
[M::main::0.003*1.20] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*1.19] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.18] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.172*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922155a1066/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922155a1066/rps24.fa /tmp/Rtmp8hsqBt/file7922155a1066/fastq/sample2.fq.gz
[M::main] Real time: 0.173 sec; CPU: 0.173 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample sample3 -> /tmp/Rtmp8hsqBt/file7922155a1066/sample3_align2genome.bam
[M::mm_idx_gen::0.001*1.33] collected minimizers
[M::mm_idx_gen::0.002*1.21] sorted minimizers
[M::main::0.002*1.21] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.20] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.19] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.330*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922155a1066/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922155a1066/rps24.fa /tmp/Rtmp8hsqBt/file7922155a1066/fastq/sample3.fq.gz
[M::main] Real time: 0.331 sec; CPU: 0.331 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: isoform_identification @ Tue Aug 12 01:28:32 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:28:33 2025 -------------------
Realigning sample sample1 -> /tmp/Rtmp8hsqBt/file7922155a1066/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.40] collected minimizers
[M::mm_idx_gen::0.002*1.31] sorted minimizers
[M::main::0.002*1.31] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.29] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.28] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.092*1.01] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922155a1066/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922155a1066/fastq/sample1.fq.gz
[M::main] Real time: 0.093 sec; CPU: 0.093 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample2 -> /tmp/Rtmp8hsqBt/file7922155a1066/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.17] collected minimizers
[M::mm_idx_gen::0.002*1.14] sorted minimizers
[M::main::0.002*1.14] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.002*1.13] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.002*1.12] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.092*1.00] mapped 100 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922155a1066/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922155a1066/fastq/sample2.fq.gz
[M::main] Real time: 0.092 sec; CPU: 0.093 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample sample3 -> /tmp/Rtmp8hsqBt/file7922155a1066/sample3_realign2transcript.bam
[M::mm_idx_gen::0.002*0.92] collected minimizers
[M::mm_idx_gen::0.003*0.93] sorted minimizers
[M::main::0.003*0.93] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*0.93] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*0.93] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.175*1.00] mapped 193 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922155a1066/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922155a1066/fastq/sample3.fq.gz
[M::main] Real time: 0.175 sec; CPU: 0.175 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Aug 12 01:28:33 2025 ----------
01:28:33 Tue Aug 12 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/Rtmp8hsqBt/file7922143f4cd37/config_file_496161.json 
Configured steps: 
	genome_alignment: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: genome_alignment @ Tue Aug 12 01:28:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
sample1 ->/tmp/Rtmp8hsqBt/file7922143f4cd37/sample1_align2genome.bam
sample2 ->/tmp/Rtmp8hsqBt/file7922143f4cd37/sample2_align2genome.bam
sample3 ->/tmp/Rtmp8hsqBt/file7922143f4cd37/sample3_align2genome.bam
-- Running step: isoform_identification @ Tue Aug 12 01:28:56 2025 -------------
Inputs:  ['/tmp/Rtmp8hsqBt/file7922155a1066/sample1_realign2transcript.bam', '/tmp/Rtmp8hsqBt/file7922155a1066/sample2_realign2transcript.bam', '/tmp/Rtmp8hsqBt/file7922155a1066/sample3_realign2transcript.bam'] /tmp/Rtmp8hsqBt/file7922155a1066/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
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:28:56 2025 -------------------
Realignment complete for the following samples:
sample1 ->/tmp/Rtmp8hsqBt/file7922143f4cd37/sample1_realign2transcript.bam
sample2 ->/tmp/Rtmp8hsqBt/file7922143f4cd37/sample2_realign2transcript.bam
sample3 ->/tmp/Rtmp8hsqBt/file7922143f4cd37/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:29:19 2025 ----------
01:29:19 Tue Aug 12 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/Rtmp8hsqBt/file7922137a1494e/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:29:20 2025 ----------------
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/Rtmp8hsqBt/file7922137a1494e/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!
-- Running step: genome_alignment @ Tue Aug 12 01:29:20 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp8hsqBt/file7922137a1494e/matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922137a1494e/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.
[M::mm_idx_gen::0.001*1.73] collected minimizers
[M::mm_idx_gen::0.002*3.49] sorted minimizers
[M::main::0.002*3.46] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*3.30] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*3.19] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.114*7.01] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/Rtmp8hsqBt/file7922137a1494e/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922137a1494e/rps24.fa /tmp/Rtmp8hsqBt/file7922137a1494e/matched_reads.fastq.gz
[M::main] Real time: 0.115 sec; CPU: 0.801 sec; Peak RSS: 0.020 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Aug 12 01:29:21 2025 ----------------
01:29:21 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file7922137a1494e/align2genome.bam'
Inputs:  ['/tmp/Rtmp8hsqBt/file7922143f4cd37/sample1_realign2transcript.bam', '/tmp/Rtmp8hsqBt/file7922143f4cd37/sample2_realign2transcript.bam', '/tmp/Rtmp8hsqBt/file7922143f4cd37/sample3_realign2transcript.bam'] /tmp/Rtmp8hsqBt/file7922143f4cd37/transcript_assembly.fa.fai 5 0.4 0.4
	Counter({'counted_reads': 391, 'not_enough_coverage': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.19gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.19gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 37013.18Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:29:22 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 ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:29:32 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/Rtmp8hsqBt/file7922137a1494e/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922137a1494e/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp8hsqBt/file7922137a1494e/matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922137a1494e/realign2transcript.bam
[M::mm_idx_gen::0.001*1.63] collected minimizers
[M::mm_idx_gen::0.002*3.27] sorted minimizers
[M::main::0.002*3.24] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*3.17] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*3.10] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.054*6.43] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 8 --seed 2022 -y /tmp/Rtmp8hsqBt/file7922137a1494e/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922137a1494e/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.055 sec; CPU: 0.351 sec; Peak RSS: 0.008 GB
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Tue Aug 12 01:29:32 2025 ----------
2025-08-12T05:29:32.953347Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:29:32.953787Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922137a1494e/realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:29:32.953797Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:29:32.953800Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:29:32.953855Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:29:32.953860Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:29:32.960548Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792215598de7/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:29:34 2025 ----------------
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/Rtmp8hsqBt/file792215598de7/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!
-- Running step: genome_alignment @ Tue Aug 12 01:29:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792215598de7/matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file792215598de7/align2genome.bam
-- Running step: gene_quantification @ Tue Aug 12 01:29:58 2025 ----------------
01:29:58 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file792215598de7/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.68gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.68gene_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, 38777.84Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:29:59 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 ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:30:09 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/Rtmp8hsqBt/file792215598de7/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792215598de7/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792215598de7/matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file792215598de7/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:30:30 2025 ----------
2025-08-12T05:30:30.170227Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:30:30.170671Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file792215598de7/realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:30:30.170684Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:30:30.170688Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:30:30.170747Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:30:30.170753Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:30:30.176946Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792213fa38c52/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:30:31 2025 ----------------
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/Rtmp8hsqBt/file792213fa38c52/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!
-- Running step: genome_alignment @ Tue Aug 12 01:30:32 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp8hsqBt/file792213fa38c52/matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file792213fa38c52/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.
[M::mm_idx_gen::0.001*1.58] collected minimizers
[M::mm_idx_gen::0.002*3.56] sorted minimizers
[M::main::0.002*3.54] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*3.38] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*3.26] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.108*7.31] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/Rtmp8hsqBt/file792213fa38c52/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file792213fa38c52/rps24.fa /tmp/Rtmp8hsqBt/file792213fa38c52/matched_reads.fastq.gz
[M::main] Real time: 0.109 sec; CPU: 0.790 sec; Peak RSS: 0.018 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Aug 12 01:30:32 2025 ----------------
01:30:32 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file792213fa38c52/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.02gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.01gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38777.55Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:30:33 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 ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:30:43 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/Rtmp8hsqBt/file792213fa38c52/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792213fa38c52/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp8hsqBt/file792213fa38c52/matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file792213fa38c52/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.
[M::mm_idx_gen::0.001*1.28] collected minimizers
[M::mm_idx_gen::0.002*2.49] sorted minimizers
[M::main::0.002*2.47] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*2.42] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*2.37] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.042*6.32] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 8 --seed 2022 /tmp/Rtmp8hsqBt/file792213fa38c52/transcript_assembly.fa /tmp/Rtmp8hsqBt/file792213fa38c52/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.043 sec; CPU: 0.267 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Tue Aug 12 01:30:43 2025 ----------
01:30:43 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792214f1615cc/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:30:44 2025 ----------------
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/Rtmp8hsqBt/file792214f1615cc/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!
-- Running step: genome_alignment @ Tue Aug 12 01:30:44 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792214f1615cc/matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file792214f1615cc/align2genome.bam
-- Running step: gene_quantification @ Tue Aug 12 01:31:04 2025 ----------------
01:31:04 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file792214f1615cc/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.90gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  1.90gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39565.17Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:31:05 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 ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:31:15 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/Rtmp8hsqBt/file792214f1615cc/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792214f1615cc/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792214f1615cc/matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file792214f1615cc/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:31:36 2025 ----------
01:31:36 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file7922135d96e87/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:31:37 2025 ----------------
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/Rtmp8hsqBt/file7922135d96e87/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!
-- Running step: genome_alignment @ Tue Aug 12 01:31:37 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp8hsqBt/file7922135d96e87/matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922135d96e87/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.
[M::mm_idx_gen::0.001*1.56] collected minimizers
[M::mm_idx_gen::0.002*1.49] sorted minimizers
[M::main::0.002*1.49] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.46] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.44] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.098*7.01] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/Rtmp8hsqBt/file7922135d96e87/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922135d96e87/rps24.fa /tmp/Rtmp8hsqBt/file7922135d96e87/matched_reads.fastq.gz
[M::main] Real time: 0.098 sec; CPU: 0.685 sec; Peak RSS: 0.019 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Aug 12 01:31:38 2025 ----------------
01:31:38 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file7922135d96e87/align2genome.bam'
	Counter({'counted_reads': 347, 'not_enough_coverage': 9, 'unmapped': 2})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.17gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.17gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39169.96Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:31:38 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:31:39 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/Rtmp8hsqBt/file7922135d96e87/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922135d96e87/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp8hsqBt/file7922135d96e87/matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922135d96e87/realign2transcript.bam
[M::mm_idx_gen::0.003*1.37] collected minimizers
[M::mm_idx_gen::0.004*1.70] sorted minimizers
[M::main::0.004*1.70] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.004*1.68] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.004*1.66] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.103*6.64] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 8 --seed 2022 -y /tmp/Rtmp8hsqBt/file7922135d96e87/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922135d96e87/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.104 sec; CPU: 0.688 sec; Peak RSS: 0.009 GB
Sorting BAM files by 8 with CB threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
-- Running step: transcript_quantification @ Tue Aug 12 01:31:40 2025 ----------
2025-08-12T05:31:40.078665Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:31:40.079203Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922135d96e87/realign2transcript.bam, contains 10 reference sequences.
2025-08-12T05:31:40.079211Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:31:40.079214Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:31:40.079277Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:31:40.079284Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-12T05:31:40.089694Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792212f5dc026/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:31:41 2025 ----------------
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/Rtmp8hsqBt/file792212f5dc026/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!
-- Running step: genome_alignment @ Tue Aug 12 01:31:42 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792212f5dc026/matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file792212f5dc026/align2genome.bam
-- Running step: gene_quantification @ Tue Aug 12 01:32:02 2025 ----------------
01:32:02 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file792212f5dc026/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.18gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.18gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38659.89Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:32:03 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:32:04 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/Rtmp8hsqBt/file792212f5dc026/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792212f5dc026/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792212f5dc026/matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file792212f5dc026/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:32:25 2025 ----------
2025-08-12T05:32:25.656864Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:32:25.657372Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file792212f5dc026/realign2transcript.bam, contains 10 reference sequences.
2025-08-12T05:32:25.657384Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:32:25.657388Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:32:25.657470Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:32:25.657478Z  INFO oarfish: parsed reference information for 10 transcripts.
2025-08-12T05:32:25.668147Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file7922140a5dcaf/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:32:27 2025 ----------------
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/Rtmp8hsqBt/file7922140a5dcaf/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!
-- Running step: genome_alignment @ Tue Aug 12 01:32:27 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp8hsqBt/file7922140a5dcaf/matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922140a5dcaf/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.
[M::mm_idx_gen::0.001*0.94] collected minimizers
[M::mm_idx_gen::0.002*0.72] sorted minimizers
[M::main::0.002*0.72] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*0.74] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*0.76] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.108*7.24] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 8 --seed 2022 --junc-bed /tmp/Rtmp8hsqBt/file7922140a5dcaf/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922140a5dcaf/rps24.fa /tmp/Rtmp8hsqBt/file7922140a5dcaf/matched_reads.fastq.gz
[M::main] Real time: 0.109 sec; CPU: 0.784 sec; Peak RSS: 0.020 GB
Sorting BAM files by genome coordinates with 8 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Aug 12 01:32:28 2025 ----------------
01:32:28 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file7922140a5dcaf/align2genome.bam'
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.09gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.09gene_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, 38589.46Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:32:28 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:32:29 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/Rtmp8hsqBt/file7922140a5dcaf/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922140a5dcaf/matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp8hsqBt/file7922140a5dcaf/matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922140a5dcaf/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.
[M::mm_idx_gen::0.002*1.85] collected minimizers
[M::mm_idx_gen::0.003*2.79] sorted minimizers
[M::main::0.003*2.77] loaded/built the index for 10 target sequence(s)
[M::mm_mapopt_update::0.003*2.71] mid_occ = 11
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 10
[M::mm_idx_stat::0.003*2.66] distinct minimizers: 611 (72.01% are singletons); average occurrences: 2.597; average spacing: 5.171; total length: 8207
[M::worker_pipeline::0.053*6.48] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 8 --seed 2022 /tmp/Rtmp8hsqBt/file7922140a5dcaf/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922140a5dcaf/matched_reads_dedup.fastq.gz
[M::main] Real time: 0.054 sec; CPU: 0.346 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 8 threads...

[bam_sort_core] merging from 0 files and 8 in-memory blocks...
Indexing bam files
-- Running step: transcript_quantification @ Tue Aug 12 01:32:29 2025 ----------
01:32:29 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(SingleCellPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792213d5cb4c3/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:32:30 2025 ----------------
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/Rtmp8hsqBt/file792213d5cb4c3/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!
-- Running step: genome_alignment @ Tue Aug 12 01:32:31 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792213d5cb4c3/matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file792213d5cb4c3/align2genome.bam
-- Running step: gene_quantification @ Tue Aug 12 01:32:53 2025 ----------------
01:32:53 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file792213d5cb4c3/align2genome.bam'
	Counter({'counted_reads': 358})
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.27gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00,  2.27gene_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, 38828.09Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:32:54 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:32:54 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/Rtmp8hsqBt/file792213d5cb4c3/matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792213d5cb4c3/matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792213d5cb4c3/matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file792213d5cb4c3/realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:33:15 2025 ----------
01:33:15 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	realign2transcript.bam
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file79221ea974f6/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:33:16 2025 ----------------
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/Rtmp8hsqBt/file79221ea974f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file79221ea974f6/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file79221ea974f6/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file79221ea974f6/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!
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/Rtmp8hsqBt/file79221ea974f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file79221ea974f6/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!
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/Rtmp8hsqBt/file79221ea974f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file79221ea974f6/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!
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/Rtmp8hsqBt/file79221ea974f6/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file79221ea974f6/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!
-- Running step: genome_alignment @ Tue Aug 12 01:33:17 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file79221ea974f6/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.
[M::mm_idx_gen::0.003*1.17] collected minimizers
[M::mm_idx_gen::0.004*1.13] sorted minimizers
[M::main::0.004*1.13] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*1.13] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.13] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.602*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file79221ea974f6/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file79221ea974f6/rps24.fa /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.602 sec; CPU: 0.603 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file79221ea974f6/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.
[M::mm_idx_gen::0.001*1.46] collected minimizers
[M::mm_idx_gen::0.002*1.31] sorted minimizers
[M::main::0.002*1.30] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.29] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.27] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.154*1.00] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file79221ea974f6/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file79221ea974f6/rps24.fa /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.154 sec; CPU: 0.155 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file79221ea974f6/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.
[M::mm_idx_gen::0.001*1.68] collected minimizers
[M::mm_idx_gen::0.002*1.44] sorted minimizers
[M::main::0.002*1.43] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.41] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.38] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.156*1.01] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file79221ea974f6/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file79221ea974f6/rps24.fa /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.156 sec; CPU: 0.157 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file79221ea974f6/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.
[M::mm_idx_gen::0.002*1.42] collected minimizers
[M::mm_idx_gen::0.002*1.29] sorted minimizers
[M::main::0.002*1.29] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.28] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.26] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.298*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file79221ea974f6/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file79221ea974f6/rps24.fa /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.298 sec; CPU: 0.299 sec; Peak RSS: 0.007 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Aug 12 01:33:19 2025 ----------------
01:33:19 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_align2genome.bam',
'/tmp/Rtmp8hsqBt/file79221ea974f6/sample1_align2genome.bam',
'/tmp/Rtmp8hsqBt/file79221ea974f6/sample2_align2genome.bam', and
'/tmp/Rtmp8hsqBt/file79221ea974f6/sample3_align2genome.bam'
	Counter({'counted_reads': 358})
parsing /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 21.52gene_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, 39426.52Read/s]
parsing /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.97gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 186281.04Read/s]
parsing /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_align2genome.bam...
Assigning reads to genes...

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

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 150752.77Read/s]
parsing /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.67gene_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, 77113.65Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:33:19 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:33:42 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file79221ea974f6/fastq, /tmp/Rtmp8hsqBt/file79221ea974f6/fastq/sample1.fq.gz, /tmp/Rtmp8hsqBt/file79221ea974f6/fastq/sample2.fq.gz, /tmp/Rtmp8hsqBt/file79221ea974f6/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_realign2transcript.bam
[M::mm_idx_gen::0.002*1.46] collected minimizers
[M::mm_idx_gen::0.002*1.40] sorted minimizers
[M::main::0.002*1.39] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.38] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.37] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.271*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp8hsqBt/file79221ea974f6/transcript_assembly.fa /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.271 sec; CPU: 0.271 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*2.50] collected minimizers
[M::mm_idx_gen::0.001*2.22] sorted minimizers
[M::main::0.001*2.20] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*2.15] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*2.11] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.071*1.02] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp8hsqBt/file79221ea974f6/transcript_assembly.fa /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.072 sec; CPU: 0.073 sec; Peak RSS: 0.003 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_realign2transcript.bam
[M::mm_idx_gen::0.002*1.28] collected minimizers
[M::mm_idx_gen::0.002*1.25] sorted minimizers
[M::main::0.002*1.25] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.002*1.24] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.23] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.082*1.01] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp8hsqBt/file79221ea974f6/transcript_assembly.fa /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.082 sec; CPU: 0.083 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.20] collected minimizers
[M::mm_idx_gen::0.001*1.15] sorted minimizers
[M::main::0.001*1.14] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.14] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.14] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.137*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp8hsqBt/file79221ea974f6/transcript_assembly.fa /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.137 sec; CPU: 0.137 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Tue Aug 12 01:33:43 2025 ----------
2025-08-12T05:33:43.310085Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:33:43.310576Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221ea974f6/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:33:43.310584Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:33:43.310587Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:33:43.310664Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:33:43.310673Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:33:43.316534Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:33:44.717921Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:33:44.718485Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221ea974f6/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:33:44.718497Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:33:44.718501Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:33:44.718558Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:33:44.718564Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:33:46.069383Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:33:46.069802Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221ea974f6/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:33:46.069815Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:33:46.069820Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:33:46.069889Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:33:46.069895Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:33:47.395926Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:33:47.396319Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221ea974f6/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:33:47.396330Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:33:47.396334Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:33:47.396389Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:33:47.396503Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file7922179991bb8/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:33:49 2025 ----------------
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/Rtmp8hsqBt/file7922179991bb8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922179991bb8/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922179991bb8/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922179991bb8/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!
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/Rtmp8hsqBt/file7922179991bb8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922179991bb8/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!
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/Rtmp8hsqBt/file7922179991bb8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922179991bb8/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!
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/Rtmp8hsqBt/file7922179991bb8/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922179991bb8/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!
-- Running step: genome_alignment @ Tue Aug 12 01:33:49 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_align2genome.bam
/tmp/Rtmp8hsqBt/file7922179991bb8/sample1_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file7922179991bb8/sample1_align2genome.bam
/tmp/Rtmp8hsqBt/file7922179991bb8/sample2_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file7922179991bb8/sample2_align2genome.bam
/tmp/Rtmp8hsqBt/file7922179991bb8/sample3_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file7922179991bb8/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Aug 12 01:34:12 2025 ----------------
01:34:12 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_align2genome.bam',
'/tmp/Rtmp8hsqBt/file7922179991bb8/sample1_align2genome.bam',
'/tmp/Rtmp8hsqBt/file7922179991bb8/sample2_align2genome.bam', and
'/tmp/Rtmp8hsqBt/file7922179991bb8/sample3_align2genome.bam'
parsing /tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.44gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39010.98Read/s]
parsing /tmp/Rtmp8hsqBt/file7922179991bb8/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.60gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 185329.54Read/s]
parsing /tmp/Rtmp8hsqBt/file7922179991bb8/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 50.31gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 154422.63Read/s]
parsing /tmp/Rtmp8hsqBt/file7922179991bb8/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.38gene_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, 77853.96Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:34:13 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:34:37 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922179991bb8/fastq, /tmp/Rtmp8hsqBt/file7922179991bb8/fastq/sample1.fq.gz, /tmp/Rtmp8hsqBt/file7922179991bb8/fastq/sample2.fq.gz, /tmp/Rtmp8hsqBt/file7922179991bb8/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922179991bb8/sample1_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922179991bb8/sample2_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922179991bb8/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922179991bb8/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922179991bb8/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922179991bb8/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_realign2transcript.bam
/tmp/Rtmp8hsqBt/file7922179991bb8/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file7922179991bb8/sample1_realign2transcript.bam
/tmp/Rtmp8hsqBt/file7922179991bb8/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file7922179991bb8/sample2_realign2transcript.bam
/tmp/Rtmp8hsqBt/file7922179991bb8/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file7922179991bb8/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:34:59 2025 ----------
2025-08-12T05:34:59.260725Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:34:59.261202Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922179991bb8/sampleA_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:34:59.261212Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:34:59.261216Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:34:59.261267Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:34:59.261274Z  INFO oarfish: parsed reference information for 5 transcripts.
2025-08-12T05:34:59.267688Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:35:00.745528Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:35:00.745916Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922179991bb8/sample1_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:35:00.745929Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:35:00.745932Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:35:00.746003Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:35:00.746010Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:35:02.109161Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:35:02.109781Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922179991bb8/sample2_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:35:02.109792Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:35:02.109795Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:35:02.109847Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:35:02.109853Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:35:03.437306Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:35:03.437855Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922179991bb8/sample3_realign2transcript.bam, contains 5 reference sequences.
2025-08-12T05:35:03.437867Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:35:03.437871Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:35:03.437927Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:35:03.437933Z  INFO oarfish: parsed reference information for 5 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792212361801b/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:35:04 2025 ----------------
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/Rtmp8hsqBt/file792212361801b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file792212361801b/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file792212361801b/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file792212361801b/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!
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/Rtmp8hsqBt/file792212361801b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file792212361801b/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!
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/Rtmp8hsqBt/file792212361801b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file792212361801b/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!
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/Rtmp8hsqBt/file792212361801b/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file792212361801b/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!
-- Running step: genome_alignment @ Tue Aug 12 01:35:05 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp8hsqBt/file792212361801b/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file792212361801b/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.
[M::mm_idx_gen::0.003*1.30] collected minimizers
[M::mm_idx_gen::0.003*1.25] sorted minimizers
[M::main::0.003*1.24] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*1.23] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.23] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.600*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file792212361801b/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file792212361801b/rps24.fa /tmp/Rtmp8hsqBt/file792212361801b/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.601 sec; CPU: 0.601 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file792212361801b/sample1_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file792212361801b/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.
[M::mm_idx_gen::0.001*1.34] collected minimizers
[M::mm_idx_gen::0.002*1.23] sorted minimizers
[M::main::0.002*1.23] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.21] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.20] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.153*1.00] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file792212361801b/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file792212361801b/rps24.fa /tmp/Rtmp8hsqBt/file792212361801b/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.154 sec; CPU: 0.155 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file792212361801b/sample2_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file792212361801b/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.
[M::mm_idx_gen::0.001*1.52] collected minimizers
[M::mm_idx_gen::0.002*1.35] sorted minimizers
[M::main::0.002*1.35] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.33] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.31] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.157*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file792212361801b/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file792212361801b/rps24.fa /tmp/Rtmp8hsqBt/file792212361801b/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.158 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file792212361801b/sample3_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file792212361801b/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.
[M::mm_idx_gen::0.002*0.97] collected minimizers
[M::mm_idx_gen::0.003*0.98] sorted minimizers
[M::main::0.003*0.98] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*0.98] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*0.98] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.299*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file792212361801b/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file792212361801b/rps24.fa /tmp/Rtmp8hsqBt/file792212361801b/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.300 sec; CPU: 0.300 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Aug 12 01:35:07 2025 ----------------
01:35:07 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file792212361801b/sampleA_align2genome.bam',
'/tmp/Rtmp8hsqBt/file792212361801b/sample1_align2genome.bam',
'/tmp/Rtmp8hsqBt/file792212361801b/sample2_align2genome.bam', and
'/tmp/Rtmp8hsqBt/file792212361801b/sample3_align2genome.bam'
parsing /tmp/Rtmp8hsqBt/file792212361801b/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.85gene_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, 39099.85Read/s]
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.52gene_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, 186244.65Read/s]
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.41gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 154102.64Read/s]
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 34.71gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77390.25Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:35:08 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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

Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:35:31 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792212361801b/fastq, /tmp/Rtmp8hsqBt/file792212361801b/fastq/sample1.fq.gz, /tmp/Rtmp8hsqBt/file792212361801b/fastq/sample2.fq.gz, /tmp/Rtmp8hsqBt/file792212361801b/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792212361801b/sampleA_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file792212361801b/sample1_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file792212361801b/sample2_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file792212361801b/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792212361801b/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file792212361801b/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file792212361801b/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file792212361801b/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp8hsqBt/file792212361801b/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file792212361801b/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.
[M::mm_idx_gen::0.001*1.73] collected minimizers
[M::mm_idx_gen::0.001*1.58] sorted minimizers
[M::main::0.001*1.58] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.55] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.53] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.229*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file792212361801b/transcript_assembly.fa /tmp/Rtmp8hsqBt/file792212361801b/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.229 sec; CPU: 0.230 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp8hsqBt/file792212361801b/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file792212361801b/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.
[M::mm_idx_gen::0.001*1.40] collected minimizers
[M::mm_idx_gen::0.001*1.31] sorted minimizers
[M::main::0.001*1.31] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.30] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.002*1.29] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.060*1.01] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file792212361801b/transcript_assembly.fa /tmp/Rtmp8hsqBt/file792212361801b/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.061 sec; CPU: 0.061 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp8hsqBt/file792212361801b/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file792212361801b/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.
[M::mm_idx_gen::0.001*2.33] collected minimizers
[M::mm_idx_gen::0.001*2.05] sorted minimizers
[M::main::0.001*2.04] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.99] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.95] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.063*1.02] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file792212361801b/transcript_assembly.fa /tmp/Rtmp8hsqBt/file792212361801b/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.064 sec; CPU: 0.065 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp8hsqBt/file792212361801b/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file792212361801b/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.
[M::mm_idx_gen::0.001*1.31] collected minimizers
[M::mm_idx_gen::0.001*1.25] sorted minimizers
[M::main::0.001*1.24] loaded/built the index for 5 target sequence(s)
[M::mm_mapopt_update::0.001*1.23] mid_occ = 10
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 5
[M::mm_idx_stat::0.001*1.22] distinct minimizers: 198 (33.33% are singletons); average occurrences: 2.884; average spacing: 5.196; total length: 2967
[M::worker_pipeline::0.116*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file792212361801b/transcript_assembly.fa /tmp/Rtmp8hsqBt/file792212361801b/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.116 sec; CPU: 0.117 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Aug 12 01:35:31 2025 ----------
01:35:31 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp8hsqBt/file792212361801b/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file792212361801b/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file792212361801b/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample1_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file792212361801b/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample2_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file792212361801b/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample3_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file792212361801b/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file792212361801b/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=TRUE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file792215e9e764/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:35:34 2025 ----------------
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/Rtmp8hsqBt/file792215e9e764/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file792215e9e764/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file792215e9e764/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file792215e9e764/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!
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/Rtmp8hsqBt/file792215e9e764/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file792215e9e764/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!
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/Rtmp8hsqBt/file792215e9e764/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file792215e9e764/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!
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/Rtmp8hsqBt/file792215e9e764/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file792215e9e764/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!
-- Running step: genome_alignment @ Tue Aug 12 01:35:34 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792215e9e764/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file792215e9e764/sampleA_align2genome.bam
/tmp/Rtmp8hsqBt/file792215e9e764/sample1_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file792215e9e764/sample1_align2genome.bam
/tmp/Rtmp8hsqBt/file792215e9e764/sample2_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file792215e9e764/sample2_align2genome.bam
/tmp/Rtmp8hsqBt/file792215e9e764/sample3_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file792215e9e764/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Aug 12 01:35:57 2025 ----------------
01:35:57 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file792215e9e764/sampleA_align2genome.bam',
'/tmp/Rtmp8hsqBt/file792215e9e764/sample1_align2genome.bam',
'/tmp/Rtmp8hsqBt/file792215e9e764/sample2_align2genome.bam', and
'/tmp/Rtmp8hsqBt/file792215e9e764/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/Rtmp8hsqBt/file792215e9e764/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 22.40gene_group/s]
/home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/python/count_gene.py:704: 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, 39335.86Read/s]
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.96gene_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, 183061.45Read/s]
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.88gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 155991.67Read/s]
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 37.08gene_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, 78196.50Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:35:58 2025 -------------
Unzipping annotation file for bambu
--- Start generating read class files ---

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Detected 6 warnings across the samples during read class construction. Access warnings with metadata(bambuOutput)$warnings
--- Start extending annotations ---
WARNING - Less than 50 TRUE or FALSE read classes for NDR precision stabilization.
NDR will be approximated as: (1 - Transcript Model Prediction Score)
For your combination of sample and reference annotations we recommend an NDR of 0. You are currently using an NDR threshold of 0.5. A higher NDR is suited for samples where the reference annotations are poor and more novel transcripts are expected,whereas a lower NDR is suited for samples with already high quality annotations
--- Start isoform quantification ---
--- Finished running Bambu ---
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:36:21 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792215e9e764/fastq, /tmp/Rtmp8hsqBt/file792215e9e764/fastq/sample1.fq.gz, /tmp/Rtmp8hsqBt/file792215e9e764/fastq/sample2.fq.gz, /tmp/Rtmp8hsqBt/file792215e9e764/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792215e9e764/sampleA_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file792215e9e764/sample1_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file792215e9e764/sample2_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file792215e9e764/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file792215e9e764/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file792215e9e764/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file792215e9e764/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file792215e9e764/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp8hsqBt/file792215e9e764/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file792215e9e764/sampleA_realign2transcript.bam
/tmp/Rtmp8hsqBt/file792215e9e764/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file792215e9e764/sample1_realign2transcript.bam
/tmp/Rtmp8hsqBt/file792215e9e764/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file792215e9e764/sample2_realign2transcript.bam
/tmp/Rtmp8hsqBt/file792215e9e764/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file792215e9e764/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:36:43 2025 ----------
01:36:44 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file792215e9e764/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample1_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file792215e9e764/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample2_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file792215e9e764/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample3_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file792215e9e764/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file792215e9e764/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file7922142fe99f7/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:36:47 2025 ----------------
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/Rtmp8hsqBt/file7922142fe99f7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922142fe99f7/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922142fe99f7/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922142fe99f7/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!
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/Rtmp8hsqBt/file7922142fe99f7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922142fe99f7/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!
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/Rtmp8hsqBt/file7922142fe99f7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922142fe99f7/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!
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/Rtmp8hsqBt/file7922142fe99f7/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922142fe99f7/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!
-- Running step: genome_alignment @ Tue Aug 12 01:36:48 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922142fe99f7/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.
[M::mm_idx_gen::0.001*1.44] collected minimizers
[M::mm_idx_gen::0.002*1.28] sorted minimizers
[M::main::0.002*1.28] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.26] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.25] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.600*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922142fe99f7/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922142fe99f7/rps24.fa /tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.601 sec; CPU: 0.601 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922142fe99f7/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.
[M::mm_idx_gen::0.001*2.02] collected minimizers
[M::mm_idx_gen::0.002*1.63] sorted minimizers
[M::main::0.002*1.63] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.60] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.57] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.152*1.01] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922142fe99f7/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922142fe99f7/rps24.fa /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.153 sec; CPU: 0.154 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922142fe99f7/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.
[M::mm_idx_gen::0.003*1.16] collected minimizers
[M::mm_idx_gen::0.003*1.13] sorted minimizers
[M::main::0.003*1.13] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.13] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.12] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.158*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922142fe99f7/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922142fe99f7/rps24.fa /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.158 sec; CPU: 0.158 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922142fe99f7/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.
[M::mm_idx_gen::0.003*1.23] collected minimizers
[M::mm_idx_gen::0.003*1.18] sorted minimizers
[M::main::0.003*1.18] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.004*1.17] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.17] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.299*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922142fe99f7/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922142fe99f7/rps24.fa /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.300 sec; CPU: 0.301 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Aug 12 01:36:50 2025 ----------------
01:36:50 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_align2genome.bam',
'/tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_align2genome.bam',
'/tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_align2genome.bam', and
'/tmp/Rtmp8hsqBt/file7922142fe99f7/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/Rtmp8hsqBt/file7922142fe99f7/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.23gene_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, 39054.56Read/s]
parsing /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 54.05gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 185871.59Read/s]
parsing /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.87gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153148.33Read/s]
parsing /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.99gene_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, 76474.20Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:36:51 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:36:51 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922142fe99f7/fastq, /tmp/Rtmp8hsqBt/file7922142fe99f7/fastq/sample1.fq.gz, /tmp/Rtmp8hsqBt/file7922142fe99f7/fastq/sample2.fq.gz, /tmp/Rtmp8hsqBt/file7922142fe99f7/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_realign2transcript.bam
[M::mm_idx_gen::0.002*1.56] collected minimizers
[M::mm_idx_gen::0.003*1.45] sorted minimizers
[M::main::0.003*1.45] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.003*1.44] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.003*1.42] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.728*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp8hsqBt/file7922142fe99f7/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.729 sec; CPU: 0.730 sec; Peak RSS: 0.007 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_realign2transcript.bam
[M::mm_idx_gen::0.001*1.55] collected minimizers
[M::mm_idx_gen::0.002*1.40] sorted minimizers
[M::main::0.002*1.40] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.38] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.37] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.198*1.00] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp8hsqBt/file7922142fe99f7/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.198 sec; CPU: 0.199 sec; Peak RSS: 0.004 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_realign2transcript.bam
[M::mm_idx_gen::0.001*1.72] collected minimizers
[M::mm_idx_gen::0.002*1.54] sorted minimizers
[M::main::0.002*1.54] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.51] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.49] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.201*1.00] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp8hsqBt/file7922142fe99f7/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.202 sec; CPU: 0.203 sec; Peak RSS: 0.003 GB
Sorting BAM files by 1 with CB threads...

Realigning sample /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_realign2transcript.bam
[M::mm_idx_gen::0.001*1.55] collected minimizers
[M::mm_idx_gen::0.002*1.41] sorted minimizers
[M::main::0.002*1.41] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.39] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.37] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.342*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 --eqx -N 100 -ax map-ont -t 1 --seed 2022 -y /tmp/Rtmp8hsqBt/file7922142fe99f7/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.343 sec; CPU: 0.343 sec; Peak RSS: 0.005 GB
Sorting BAM files by 1 with CB threads...

-- Running step: transcript_quantification @ Tue Aug 12 01:36:53 2025 ----------
2025-08-12T05:36:53.780379Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:36:53.781004Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922142fe99f7/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-08-12T05:36:53.781015Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:36:53.781018Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:36:53.781100Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:36:53.781107Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-12T05:36:53.793151Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:36:55.223860Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:36:55.224367Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922142fe99f7/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-08-12T05:36:55.224375Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:36:55.224378Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:36:55.224472Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:36:55.224480Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:36:56.661422Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:36:56.661822Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922142fe99f7/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-08-12T05:36:56.661834Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:36:56.661837Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:36:56.661921Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:36:56.661929Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:36:58.131045Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:36:58.131521Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file7922142fe99f7/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-08-12T05:36:58.131531Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:36:58.131534Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:36:58.131629Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:36:58.131639Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=TRUE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file79221785d3de1/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:36:59 2025 ----------------
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/Rtmp8hsqBt/file79221785d3de1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file79221785d3de1/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file79221785d3de1/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file79221785d3de1/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!
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/Rtmp8hsqBt/file79221785d3de1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file79221785d3de1/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!
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/Rtmp8hsqBt/file79221785d3de1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file79221785d3de1/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!
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/Rtmp8hsqBt/file79221785d3de1/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file79221785d3de1/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!
-- Running step: genome_alignment @ Tue Aug 12 01:37:00 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_align2genome.bam
/tmp/Rtmp8hsqBt/file79221785d3de1/sample1_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file79221785d3de1/sample1_align2genome.bam
/tmp/Rtmp8hsqBt/file79221785d3de1/sample2_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file79221785d3de1/sample2_align2genome.bam
/tmp/Rtmp8hsqBt/file79221785d3de1/sample3_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file79221785d3de1/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Aug 12 01:37:23 2025 ----------------
01:37:23 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_align2genome.bam',
'/tmp/Rtmp8hsqBt/file79221785d3de1/sample1_align2genome.bam',
'/tmp/Rtmp8hsqBt/file79221785d3de1/sample2_align2genome.bam', and
'/tmp/Rtmp8hsqBt/file79221785d3de1/sample3_align2genome.bam'
parsing /tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 15.41gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 38007.72Read/s]
parsing /tmp/Rtmp8hsqBt/file79221785d3de1/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 48.36gene_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, 182253.28Read/s]
parsing /tmp/Rtmp8hsqBt/file79221785d3de1/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 51.12gene_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, 150689.95Read/s]
parsing /tmp/Rtmp8hsqBt/file79221785d3de1/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 33.12gene_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, 76427.38Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:37:24 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:37:25 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file79221785d3de1/fastq, /tmp/Rtmp8hsqBt/file79221785d3de1/fastq/sample1.fq.gz, /tmp/Rtmp8hsqBt/file79221785d3de1/fastq/sample2.fq.gz, /tmp/Rtmp8hsqBt/file79221785d3de1/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file79221785d3de1/sample1_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file79221785d3de1/sample2_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file79221785d3de1/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file79221785d3de1/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file79221785d3de1/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file79221785d3de1/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_realign2transcript.bam
/tmp/Rtmp8hsqBt/file79221785d3de1/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file79221785d3de1/sample1_realign2transcript.bam
/tmp/Rtmp8hsqBt/file79221785d3de1/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file79221785d3de1/sample2_realign2transcript.bam
/tmp/Rtmp8hsqBt/file79221785d3de1/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file79221785d3de1/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:37:50 2025 ----------
2025-08-12T05:37:50.305392Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:37:50.305977Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221785d3de1/sampleA_realign2transcript.bam, contains 14 reference sequences.
2025-08-12T05:37:50.305990Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:37:50.305994Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:37:50.306081Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:37:50.306090Z  INFO oarfish: parsed reference information for 14 transcripts.
2025-08-12T05:37:50.318158Z  INFO oarfish::single_cell: Processed 100 cells.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:37:52.029522Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:37:52.030234Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221785d3de1/sample1_realign2transcript.bam, contains 14 reference sequences.
2025-08-12T05:37:52.030247Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:37:52.030250Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:37:52.030340Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:37:52.030348Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:37:53.537244Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:37:53.537646Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221785d3de1/sample2_realign2transcript.bam, contains 14 reference sequences.
2025-08-12T05:37:53.537658Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:37:53.537661Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:37:53.537744Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:37:53.537765Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
2025-08-12T05:37:55.021077Z  INFO oarfish: setting user-provided filter parameters.
2025-08-12T05:37:55.021450Z  INFO oarfish::alignment_parser: read header from BAM file /tmp/Rtmp8hsqBt/file79221785d3de1/sample3_realign2transcript.bam, contains 14 reference sequences.
2025-08-12T05:37:55.021458Z  INFO oarfish::alignment_parser: saw minimap2 as a program in the header; proceeding.
2025-08-12T05:37:55.021461Z  INFO oarfish::util::digest_utils: calculating seqcol digest
2025-08-12T05:37:55.021547Z  INFO oarfish::util::digest_utils: done calculating seqcol digest
2025-08-12T05:37:55.021554Z  INFO oarfish: parsed reference information for 14 transcripts.
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file7922168d45eb2/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:37:56 2025 ----------------
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/Rtmp8hsqBt/file7922168d45eb2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922168d45eb2/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922168d45eb2/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922168d45eb2/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!
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/Rtmp8hsqBt/file7922168d45eb2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922168d45eb2/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!
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/Rtmp8hsqBt/file7922168d45eb2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922168d45eb2/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!
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/Rtmp8hsqBt/file7922168d45eb2/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922168d45eb2/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!
-- Running step: genome_alignment @ Tue Aug 12 01:37:57 2025 -------------------
Creating junction bed file from GFF3 annotation.
Aligning sample /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922168d45eb2/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.
[M::mm_idx_gen::0.002*1.12] collected minimizers
[M::mm_idx_gen::0.003*1.09] sorted minimizers
[M::main::0.003*1.09] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.08] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.003*1.08] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.602*1.00] mapped 372 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922168d45eb2/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922168d45eb2/rps24.fa /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_matched_reads.fastq.gz
[M::main] Real time: 0.603 sec; CPU: 0.603 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922168d45eb2/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.
[M::mm_idx_gen::0.003*1.00] collected minimizers
[M::mm_idx_gen::0.003*1.00] sorted minimizers
[M::main::0.003*1.00] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.003*1.00] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.004*1.00] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.154*1.00] mapped 93 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922168d45eb2/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922168d45eb2/rps24.fa /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_matched_reads.fastq.gz
[M::main] Real time: 0.155 sec; CPU: 0.155 sec; Peak RSS: 0.005 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922168d45eb2/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.
[M::mm_idx_gen::0.001*1.26] collected minimizers
[M::mm_idx_gen::0.002*1.17] sorted minimizers
[M::main::0.002*1.17] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.15] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.15] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.156*1.00] mapped 96 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922168d45eb2/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922168d45eb2/rps24.fa /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_matched_reads.fastq.gz
[M::main] Real time: 0.157 sec; CPU: 0.157 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Aligning sample /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_matched_reads.fastq.gz -> /tmp/Rtmp8hsqBt/file7922168d45eb2/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.
[M::mm_idx_gen::0.001*1.22] collected minimizers
[M::mm_idx_gen::0.002*1.15] sorted minimizers
[M::main::0.002*1.15] loaded/built the index for 1 target sequence(s)
[M::mm_mapopt_update::0.002*1.14] mid_occ = 10
[M::mm_idx_stat] kmer size: 14; skip: 5; is_hpc: 0; #seq: 1
[M::mm_idx_stat::0.002*1.13] distinct minimizers: 1763 (99.72% are singletons); average occurrences: 1.005; average spacing: 2.918; total length: 5170
[mm_idx_bed_read_merge] read 93 introns, 45 of which are non-redundant
[M::worker_pipeline::0.298*1.00] mapped 183 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax splice -k14 --secondary=no -t 1 --seed 2022 --splice-flank=no --junc-bed /tmp/Rtmp8hsqBt/file7922168d45eb2/reference.bed --junc-bonus 1 /tmp/Rtmp8hsqBt/file7922168d45eb2/rps24.fa /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_matched_reads.fastq.gz
[M::main] Real time: 0.299 sec; CPU: 0.299 sec; Peak RSS: 0.006 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: gene_quantification @ Tue Aug 12 01:37:59 2025 ----------------
01:37:59 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_align2genome.bam',
'/tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_align2genome.bam',
'/tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_align2genome.bam', and
'/tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_align2genome.bam'
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 23.11gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 39231.81Read/s]
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 49.04gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 187913.48Read/s]
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.44gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 153081.26Read/s]
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 36.85gene_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, 76947.85Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:37:59 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:38:00 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922168d45eb2/fastq, /tmp/Rtmp8hsqBt/file7922168d45eb2/fastq/sample1.fq.gz, /tmp/Rtmp8hsqBt/file7922168d45eb2/fastq/sample2.fq.gz, /tmp/Rtmp8hsqBt/file7922168d45eb2/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_matched_reads_dedup.fastq.gz
	files found
Realigning sample /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922168d45eb2/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.
[M::mm_idx_gen::0.001*1.53] collected minimizers
[M::mm_idx_gen::0.002*1.42] sorted minimizers
[M::main::0.002*1.42] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.40] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.38] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.351*1.00] mapped 358 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922168d45eb2/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.351 sec; CPU: 0.352 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922168d45eb2/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.
[M::mm_idx_gen::0.001*0.87] collected minimizers
[M::mm_idx_gen::0.002*0.90] sorted minimizers
[M::main::0.002*0.90] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*0.91] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*0.91] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.093*1.00] mapped 91 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922168d45eb2/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.094 sec; CPU: 0.093 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922168d45eb2/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.
[M::mm_idx_gen::0.002*1.26] collected minimizers
[M::mm_idx_gen::0.002*1.20] sorted minimizers
[M::main::0.002*1.20] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.19] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.18] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.094*1.00] mapped 95 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922168d45eb2/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.095 sec; CPU: 0.095 sec; Peak RSS: 0.003 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
Realigning sample /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_matched_reads_dedup.fastq.gz -> /tmp/Rtmp8hsqBt/file7922168d45eb2/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.
[M::mm_idx_gen::0.001*1.36] collected minimizers
[M::mm_idx_gen::0.002*1.28] sorted minimizers
[M::main::0.002*1.28] loaded/built the index for 14 target sequence(s)
[M::mm_mapopt_update::0.002*1.27] mid_occ = 15
[M::mm_idx_stat] kmer size: 15; skip: 10; is_hpc: 0; #seq: 14
[M::mm_idx_stat::0.002*1.25] distinct minimizers: 632 (64.56% are singletons); average occurrences: 3.326; average spacing: 5.176; total length: 10880
[M::worker_pipeline::0.178*1.00] mapped 176 sequences
[M::main] Version: 2.29-r1283
[M::main] CMD: /home/biocbuild/bbs-3.22-bioc/R/site-library/FLAMES/bin/minimap2 -ax map-ont -p 0.9 --end-bonus 10 -N 3 -t 1 --seed 2022 /tmp/Rtmp8hsqBt/file7922168d45eb2/transcript_assembly.fa /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_matched_reads_dedup.fastq.gz
[M::main] Real time: 0.179 sec; CPU: 0.179 sec; Peak RSS: 0.004 GB
Sorting BAM files by genome coordinates with 1 threads...

Indexing bam files
-- Running step: transcript_quantification @ Tue Aug 12 01:38:01 2025 ----------
01:38:01 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file7922168d45eb2/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file7922168d45eb2/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file7922168d45eb2/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file7922168d45eb2/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
Testing: run_FLAMES(MultiSampleSCPipeline, bambu=FALSE, oarfish=FALSE, controller=none)
Writing configuration parameters to:  /tmp/Rtmp8hsqBt/file7922120e1b6cc/config_file_496161.json 
Configured steps: 
	barcode_demultiplex: TRUE
	genome_alignment: TRUE
	gene_quantification: TRUE
	isoform_identification: TRUE
	read_realignment: TRUE
	transcript_quantification: TRUE
samtools not found, will use Rsamtools package instead
-- Running step: barcode_demultiplex @ Tue Aug 12 01:38:04 2025 ----------------
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/Rtmp8hsqBt/file7922120e1b6cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922120e1b6cc/fastq/sample1.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922120e1b6cc/fastq/sample2.fq.gz
Searching for barcodes...
Processing file: /tmp/Rtmp8hsqBt/file7922120e1b6cc/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!
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/Rtmp8hsqBt/file7922120e1b6cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922120e1b6cc/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!
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/Rtmp8hsqBt/file7922120e1b6cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922120e1b6cc/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!
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/Rtmp8hsqBt/file7922120e1b6cc/bc_allow.tsv
Number of known barcodes: 143
Processing file: /tmp/Rtmp8hsqBt/file7922120e1b6cc/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!
-- Running step: genome_alignment @ Tue Aug 12 01:38:04 2025 -------------------
Creating junction bed file from GFF3 annotation.
Alignment complete for the following samples:
/tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_align2genome.bam
/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_align2genome.bam
/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_align2genome.bam
/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_matched_reads.fastq.gz ->/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_align2genome.bam
-- Running step: gene_quantification @ Tue Aug 12 01:38:28 2025 ----------------
01:38:28 Tue Aug 12 2025 quantify genes 
Using BAM(s): '/tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_align2genome.bam',
'/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_align2genome.bam',
'/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_align2genome.bam', and
'/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_align2genome.bam'
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 175, 'not_enough_coverage': 1})
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 13.93gene_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, 39512.54Read/s]
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 53.55gene_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, 189530.23Read/s]
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 52.49gene_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, 155704.44Read/s]
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_align2genome.bam...
Assigning reads to genes...

Processed:   0%|          | 0/1 [00:00<?, ?gene_group/s]
Processed: 100%|██████████| 1/1 [00:00<00:00, 35.77gene_group/s]
Writing the gene count matrix ...
Plotting the saturation curve ...
Generating deduplicated fastq file ...

Processed: 0Read [00:00, ?Read/s]
Processed: 2500.0Read [00:00, 77470.30Read/s]
-- Running step: isoform_identification @ Tue Aug 12 01:38:29 2025 -------------
#### Read gene annotations
	Removed similar transcripts in gene annotation: Counter()
#### find isoforms
chr14
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
-- Running step: read_realignment @ Tue Aug 12 01:38:29 2025 -------------------
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922120e1b6cc/fastq, /tmp/Rtmp8hsqBt/file7922120e1b6cc/fastq/sample1.fq.gz, /tmp/Rtmp8hsqBt/file7922120e1b6cc/fastq/sample2.fq.gz, /tmp/Rtmp8hsqBt/file7922120e1b6cc/fastq/sample3.fq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_matched_reads.fastq.gz, /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_matched_reads.fastq.gz
	files found
Checking for fastq file(s) /tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_matched_reads_dedup.fastq.gz, /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_matched_reads_dedup.fastq.gz
	files found
Realignment complete for the following samples:
/tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_realign2transcript.bam
/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_realign2transcript.bam
/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_realign2transcript.bam
/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_matched_reads_dedup.fastq.gz ->/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_realign2transcript.bam
-- Running step: transcript_quantification @ Tue Aug 12 01:38:51 2025 ----------
01:38:51 Tue Aug 12 2025 quantify transcripts 
Found realignment file(s): 	sample1_realign2transcript.bam
	sample2_realign2transcript.bam
	sample3_realign2transcript.bam
	sampleA_realign2transcript.bam
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file7922120e1b6cc/sampleA_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample1_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample2_realign2transcript.bamdone
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_realign2transcript.bam...
parsing /tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_realign2transcript.bamdone
wrt_tr_to_csv for/tmp/Rtmp8hsqBt/file7922120e1b6cc/sample3_realign2transcript.bamdone
annotate_full_splice_match_all_sample...
[ FAIL 0 | WARN 235 | SKIP 0 | PASS 37 ]

[ FAIL 0 | WARN 235 | SKIP 0 | PASS 37 ]
	Counter({'counted_reads': 357, 'not_enough_coverage': 1})
	Counter({'counted_reads': 91})
	Counter({'counted_reads': 95})
	Counter({'counted_reads': 175, 'not_enough_coverage': 1})
> 
> proc.time()
   user  system elapsed 
817.556  57.345 873.338