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3 hours ago by

Hello,

I am trying to check the strandedness of my RNAseq files using the how_are_we_stranded_here tool. But when I enter the command:

check_strandedness --gtf $RNA_HOME/refs/sacCer3.ncbiRefSeq.gtf --transcripts $RNA_HOME/refs/sacCer3_transcripts.fa --reads_1 $RNA_DATA_DIR/GS_UM_H_S19_L008_R1_001.fastq.gz --reads_2 $RNA_DATA_DIR/GS_UM_H_S19_L008_R2_001.fastq.gz

everything seems to process properly until it begins checking strandedness (see output below)

Results stored in: stranded_test_GS_UM_H_S19_L008_R1_001
converting gtf to bed
generating kallisto index

[build] loading fasta file /home/bioinformatics/workspace/rnaseq/refs/sacCer3_transcripts.fa
[build] k-mer length: 31
[build] counting k-mers ... done.
[build] building target de Bruijn graph ...  done 
[build] creating equivalence classes ...  done
[build] target de Bruijn graph has 11222 contigs and contains 8216987 k-mers 

creating fastq files with first 200000 reads
quantifying with kallisto

[quant] fragment length distribution will be estimated from the data
[index] k-mer length: 31
[index] number of targets: 6,125
[index] number of k-mers: 8,216,987
[index] number of equivalence classes: 7,443
Warning: 6125 transcripts were defined in GTF file, but not in the index
[quant] running in paired-end mode
[quant] will process pair 1: stranded_test_GS_UM_H_S19_L008_R1_001/GS_UM_H_S19_L008_R1_001_sample.fq
                             stranded_test_GS_UM_H_S19_L008_R1_001/GS_UM_H_S19_L008_R2_001_sample.fq
[quant] finding pseudoalignments for the reads ... done
[quant] processed 200,000 reads, 175,255 reads pseudoaligned
[quant] estimated average fragment length: 172.246
[   em] quantifying the abundances ... done
[   em] the Expectation-Maximization algorithm ran for 565 rounds
[  bam] writing pseudoalignments to BAM format .. Segmentation fault (core dumped)
checking strandedness


stranded_test_GS_UM_H_S19_L008_R1_001/kallisto_strand_test/pseudoalignments.bam does NOT exists.

Traceback (most recent call last):
  File "/home/bioinformatics/miniconda3/bin/check_strandedness", line 8, in <module>
    sys.exit(main())
  File "/home/bioinformatics/miniconda3/lib/python3.8/site-packages/how_are_we_stranded_here/check_strandedness.py", line 151, in main
    result = pd.read_csv(test_folder + '/' + 'strandedness_check.txt', sep="n", header=None)
  File "/home/bioinformatics/miniconda3/lib/python3.8/site-packages/pandas/io/parsers.py", line 686, in read_csv
    return _read(filepath_or_buffer, kwds)
  File "/home/bioinformatics/miniconda3/lib/python3.8/site-packages/pandas/io/parsers.py", line 452, in _read
    parser = TextFileReader(fp_or_buf, **kwds)
  File "/home/bioinformatics/miniconda3/lib/python3.8/site-packages/pandas/io/parsers.py", line 936, in __init__
    self._make_engine(self.engine)
  File "/home/bioinformatics/miniconda3/lib/python3.8/site-packages/pandas/io/parsers.py", line 1168, in _make_engine
    self._engine = CParserWrapper(self.f, **self.options)
  File "/home/bioinformatics/miniconda3/lib/python3.8/site-packages/pandas/io/parsers.py", line 1998, in __init__
    self._reader = parsers.TextReader(src, **kwds)
  File "pandas/_libs/parsers.pyx", line 540, in pandas._libs.parsers.TextReader.__cinit__
pandas.errors.EmptyDataError: No columns to parse from file

Thank you

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3 hours ago
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bertb0



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