Mapping quality and clipping in BWA

[WhatsOEver]

Hi @all,

I have a problem in understanding some alignments produced by BWA mem (Version: 0.7.8-r455):

IQ4WJ2H02II3G5	0	chromosome_1	256920	60	512S33M	*	0	0
IQ4WJ2H02GYKYP	0	chromosome_1	380024	12	312S33M142S	*	0	0
IQ4WJ2H01BLH7R	16	chromosome_1	794344	7	79M178S	*	0	0

1) Besides the obvious (all alignments are crap)... is MapQ influenced by clipping at all? Why not? What about other aligners?

2) Next question would be if it is possible to prevent things like this from happening? I tried to adjust the "-L" parameter but it seems to have almost no effect - I tried standard (5), 50, 100, 10000 and these alignments occur in all 4 runs.

3) Has anyone of you ever seen something like this before? How do you handle/filter these reads then? The only thing that occurs to me is selecting by S/H with values greater than 50(?),100(?),10% read length(?)

Any suggestion helps!

[Brian Bushnell]

1) BBMap's map scores are not directly affected by the bases in the clipped portion, though they are affected by the length of the aligned portion, so 100= would score slightly higher than 50=50S. Also, shorter alignments are more likely to be coincidentally ambiguous so they will tend to have a slightly lower score. That said, clipping is disabled by default as BBMap is a global aligner. For BWA, I don't know how the score is calculated.

3) I've seen alignments like that when mapping to the wrong reference (e.g. contaminant reads). You might want to gather some of those mostly-clipped reads and blast them to nt; that may give you insight into how to filter them. Also, if a read starts out normal then becomes junk (for example, short-insert read that hits adapter sequence and then random letters, or a PacBio read where the enzyme breaks down partway through) you get those local alignments with most of the read clipped.

[dpryan]

1. Not directly, see a discussion here.
2. Maybe decrease -B, though I worry that this will just decrease the reliability of alignments. It'd be better to just blast things.
3. Nope, as Brian suggested, I'd blast the soft-clipped portions of a few reads.