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Old 10-10-2018, 02:16 AM   #1
Hyzteria
Junior Member
 
Location: France

Join Date: Oct 2018
Posts: 1
Default 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
OSError: [Errno 2] No such file or directory

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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