I have aligned my BS-Seq data with Bismark and I removed as well the duplicated fragments (I am following the commands from this link). My question is: do you normally filter those reads with a mapping quality, MAPQ, below a threshold in BS-Seq? Because the link provided does not say anything about it.
Thank you very much.
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int11ap1
1 answer
Yes, I personally recommend excluding alignments with MAPQ < 10. You can see why in the Bison paper and since bismark uses the same algorithm to calculate MAPQ scores (it just ported my C code to perl) you would be best off doing the same. I also exclude base calls with Phred scores < 5 or so. Presumably bismark's tools allow doing this, though if not you can always use PileOMeth, which can plot methylation bias graphs (and exclude biased regions) as well.
Bismark was modified after the paper was published (or at least submitted, I don't recall which). If you still have alignments with 255 MAPQ scores then you must be using an older version of bismark.
Ah, yeah, that would do it. Yes, use bowtie2.
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