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  • BS-seeker2 alignment error (bowtie2)

    Hi everyone ,

    I'm trying to align some of ours RRBS data on human genome hg38 with BS-seeker2.
    Indexing went good without any error except the warning :
    Code:
    Warning: Encountered reference sequence with only gaps
    but I know why.

    I get errors in the alignement step about some file or directory not found, as you can see :

    Code:
    -bash-4.1$ ./bs_seeker2-align.py -i /YYYYYYY/NNRD204.fastq.gz --aligner=bowtie2 -o PostCT_6m_bs-seeker2.bam -g ../../sequence/hg38.fa -r -L 20 -U 250 -t Y
    
         BS-Seeker2 v2.1.5 - Dec. 21, 2017
    
    [2018-10-10 11:24:42] Mode: Bowtie2, local alignment
    [2018-10-10 11:24:42] Filter for tag XS: #(mCH)/#(all CH)>50.00% and #(mCH)>5
    [2018-10-10 11:24:42] Temporary directory: /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M
    [2018-10-10 11:24:42] Reduced Representation Bisulfite Sequencing: True
    [2018-10-10 11:24:42] Single end
    [2018-10-10 11:24:42] Aligner command: None/bowtie2 --local --quiet -p 2 -D 50 --norc --sam-nohead  -k 2 -x %(reference_genome)s -f -U %(input_file)s -S %(output_file)s
    [2018-10-10 11:24:42] ----------------------------------------------
    [2018-10-10 11:24:42] Read filename: /YYYYYYYY/NNRD204.fastq.gz
    [2018-10-10 11:24:42] The first base (for mapping): 1
    [2018-10-10 11:24:42] The  last base (for mapping): 200
    [2018-10-10 11:24:42] Path for short reads aligner: None/bowtie2 --local --quiet -p 2 -D 50 --norc --sam-nohead  -k 2 -x %(reference_genome)s -f -U %(input_file)s -S %(output_file)s
    
    [2018-10-10 11:24:42] Reference genome library path: /XXXXXX/BS-seeker2/bs_utils/reference_genomes/hg38.fa_rrbs_20_250_bowtie2
    [2018-10-10 11:24:42] Un-directional library
    [2018-10-10 11:24:42] Number of mismatches allowed: 4
    [2018-10-10 11:24:42] --------------------------------
    [2018-10-10 11:24:42] Start reading and trimming the input sequences
    [2018-10-10 11:24:55] Processing read file: /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/NNRD204.fastq.gz-s-1
    [2018-10-10 11:25:09] Processing input is done
    [2018-10-10 11:25:09] Start mapping
    [2018-10-10 11:25:09] Starting commands:
    [2018-10-10 11:25:09] Launched: None/bowtie2 --local --quiet -p 2 -D 50 --norc --sam-nohead  -k 2 -x /XXXXXX/BS-seeker2/bs_utils/reference_genomes/hg38.fa_rrbs_20_250_bowtie2/W_C2T -f -U /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/Trimmed_C2T.fa.tmp-9757942 -S /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/W_C2T_m4.mapping.tmp-9757942
    [2018-10-10 11:25:09] Launched: None/bowtie2 --local --quiet -p 2 -D 50 --norc --sam-nohead  -k 2 -x /XXXXXX/BS-seeker2/bs_utils/reference_genomes/hg38.fa_rrbs_20_250_bowtie2/C_C2T -f -U /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/Trimmed_C2T.fa.tmp-9757942 -S /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/C_C2T_m4.mapping.tmp-9757942
    [2018-10-10 11:25:09] Launched: None/bowtie2 --local --quiet -p 2 -D 50 --norc --sam-nohead  -k 2 -x /XXXXXXX/BS-seeker2/bs_utils/reference_genomes/hg38.fa_rrbs_20_250_bowtie2/W_G2A -f -U /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/Trimmed_G2A.fa.tmp-9757942 -S /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/W_G2A_m4.mapping.tmp-9757942
    [2018-10-10 11:25:09] Launched: None/bowtie2 --local --quiet -p 2 -D 50 --norc --sam-nohead  -k 2 -x /XXXXXXX/BS-seeker2/bs_utils/reference_genomes/hg38.fa_rrbs_20_250_bowtie2/C_G2A -f -U /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/Trimmed_G2A.fa.tmp-9757942 -S /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/C_G2A_m4.mapping.tmp-9757942
    
    Traceback (most recent call last):
      File "./bs_seeker2-align.py", line 367, in <module>
        options.cut_format
      File "/XXXXXXX/BS-seeker2/bs_align/bs_rrbs.py", line 682, in bs_rrbs
        'output_file' : CG2A} ])
      File "/XXXXXXX/BS-seeker2/bs_utils/utils.py", line 332, in run_in_parallel
        for i, proc in enumerate([subprocess.Popen(args = shlex.split(cmd), stdout = stdout) for cmd, stdout in commands]):
      File "/local/miniconda3/envs/python-2.7.15/lib/python2.7/subprocess.py", line 394, in __init__
        errread, errwrite)
      File "/local/miniconda3/envs/python-2.7.15/lib/python2.7/subprocess.py", line 1047, in _execute_child
        raise child_exception
    [B]OSError: [Errno 2] No such file or directory[/B]

    So I tried to launch bowtie2 alignements with the commands just preciding the error (without the --quiet flag) and it went good, no error to declare :
    Code:
    bowtie2 --local -p 2 -D 50 --norc --sam-nohead  -k 2 -x /XXXXXX/BS-seeker2/bs_utils/reference_genomes/hg38.fa_rrbs_20_250_bowtie2/W_C2T -f -U /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/Trimmed_C2T.fa.tmp-9757942 -S /tmp/bs_seeker2_PostCT_6m_bs-seeker2.bam_-bowtie2-local-TMP-FAvs7M/W_C2T_m4.mapping.tmp-9757942
    I don't get why it raises me this error. I tried on local and on a distant cluster .
    I would be very grateful if someone could help me !

    Have a nice day !

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