Dear all,
After various unsuccessful attempts, and browsing the archive (partially
fixed my problem), I am afraid I have to post here for help fixing my issue.
I am trying to use CollectAlignmentSummaryMetrics for bisulfite libraries.
My problem is that adapters are not detected by Picard while I can clearly
find 3.5% of the first 1,000,000 read pairs with the first mate containing
the reverse complement of the reverse strand adapter (i.e. perfect match to
the adapter sequence detected by grep command). Maybe I am using the
ADAPTER_SEQUENCE of Picard wrong?
Similarly to the post
https://sourceforge.net/p/samtools/mailman/message/32771613/ I was getting
a lot of zeros when the reference genome sequence was not specified.
However, giving a reference genome did not improve the detection of adapter
sequences. Anything I am missing to detect the adapter sequences in my
command line below ?
Here is the command I used (in doubt, I gave the two adapter sequences +
the two reverse complement sequences, but apparently none are detected):
java -jar /usr/local/src/picard-tools-1.128/picard.jar
CollectAlignmentSummaryMetrics
INPUT=/workspace/scratch/krue/Methylation/bwa_8lanes/C12.bam
OUTPUT=/workspace/scratch/krue/Methylation/bwa_8lanes/C12.test_picard_summary.txt
ADAPTER_SEQUENCE=null ADAPTER_SEQUENCE=GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
ADAPTER_SEQUENCE=TACACTCTTTCCCTACACGACGCTCTTCCGATCT
ADAPTER_SEQUENCE=AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC
ADAPTER_SEQUENCE=AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTA
IS_BISULFITE_SEQUENCED=true
REFERENCE_SEQUENCE=/workspace/storage/genomes/bostaurus/UMD3.1.75/source_file/Bos_taurus.UMD3.1.75.dna.toplevel.fa
STOP_AFTER=100000
When I tested the SAM file, PicardValidateSamFile
<http://broadinstitute.github.io/picard/command-line-overview.html#ValidateSamFile>
returned only one error:
ERROR: Read name C12_TAGCTT_L002_R_001, A platform (PL) attribute was not
found for read group
Many thanks in advance, and I hope I didn't miss an embarrassingly obvious
explanation somewhere
Kevin
--
Kévin RUE-ALBRECHT
Wellcome Trust Computational Infection Biology PhD Programme
University College Dublin
Ireland
http://fr.linkedin.com/pub/k%C3%A9vin-rue/28/a45/149/en
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