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Cross-posted on Biostars (with no answers currently). I hope that's OK.

I thought that iff a read matches the reference at a position, it would appear in the pileup column for that position. But tracking a single read in pysam shows that that's not the case.

This read appears in some pileup columns for positions to which it does not map, and does not appear in some pileup columns to which it does map.

What's wrong in my understanding?

Updated code (and updated output) moved to Gist.

Edit: Here's the raw SAM read. I wasn't separating the read from it's mate when I calculated the figures above.

E00489:44:HNNVYCCXX:1:2120:26524:19135  163     5       345248  0       48M102S =       345446  350     TGTGTGTGTGTGTGTGTGTGTCGGATGATGTCCCTGGCTGTGTGTGGGCGGGAGTGCGTGGGGGAGGGTGAGAGTGTGGATGTCGGTGGTCGCGGCTGCGTGAGAGAGGGGGTGTGTGGGGGGGGGGGGGGGGGGGGGTGTGGGTGGGCG  AAAFFJFJJJJJJJJJJJJJJJJJJJJJJJJJFJJJJJJJJJJJ-J-7-777-7-7-7--7-77-77F---7-7AFJ7----7-7FAA7-7---7----77-777------7-7)7-AAA-A)7FA)<)<--)<)--<-FAJFJFFFF-<  XA:Z:5,+345312,48M102S,0;5,+345554,48M102S,1;5,+345452,4S44M102S,0;5,+345192,48M102S,1; MC:Z:6M2D144M   MD:Z:48 RG:Z:HNNVYCCXX.1       NM:i:0   MQ:i:60 AS:i:48 XS:i:48
E00489:44:HNNVYCCXX:1:2120:26524:19135  83      5       345446  60      6M2D144M        =       345248  -350    TGGGGATGTGTGTGTGTGTGTCGGATGATGTCCCTGGTTGTGTGTGGGGATGTGTGTGTGTGTGGGTTGATGGTCCTGGCTGTGTGTGTGTGTGGGTGTGCGTGTGTGGGTGTGTGTGTGTGTGTGTCGGATGATGTCCCTGGCTGTGTG  JJAF7-FJJJFJFF-AFJF7--<A--<F<7-7--FA--F-J--A-7--7-FJFJ7JFJJF<JA77-7----7----<F7-<JJF-<<FAJJJJA-7-<JAJJ<7FFJJ<JJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJJFFFAA  MC:Z:48M102S    MD:Z:6^TG31C25C2A5T0C76 RG:Z:HNNVYCCXX.1        NM:i:7  MQ:i:0  AS:i:119        XS:i:69

I guess this explains why the read appears in more than 150 pileup columns—in some of the columns, it's really its mate. But given the CIGAR strings for these paired reads, I would expect one of these mates to appear in 198 = (48 + 6 + 144) pileup columns, at each position where one of them maps?

Update: I re-ran this script, using @Bioathlete's suggestion of using the flags to distinguish the read and its mate. In case pileup() was filtering reads with low base quality, I also calculated how many positions the read has baseQ >= 13. It's 150 positions, but this read still appears in pileup columns for only 121 positions.

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  • $\begingroup$ Can you include the raw sam lines for the read so we can determine if the issue is in pysam or your code. That would give us truth. $\endgroup$
    – Bioathlete
    Jul 5, 2018 at 19:06
  • $\begingroup$ @Bioathlete please see above $\endgroup$
    – Randoms
    Jul 5, 2018 at 19:16

1 Answer 1

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The two entries in the sam file represent mate pairs for the given ID. You can tell the difference based on the sam flags. Picard has a nice tool to determine the meaning of the flags.

You are missing coverage within the first read. I think that it is related to the low quality bases. samtools mpileup filters bases with a quality below 13 and pysam just wraps the samtools C functions.

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  • $\begingroup$ Thanks for the tip about distinguishing a read and its mate by QNAME and flag—that's helpful. The read with flag 83, though, has baseQ > 13 at 150 positions, yet appears in the pileup columns for only 121 positions. $\endgroup$
    – Randoms
    Jul 6, 2018 at 0:53
  • $\begingroup$ Ah but BAQ ("Base Alignment Quality"), not just regular base quality, also has to be above 13. Comparing samtools mpileup and samtools mpileup -Q0 shows that that's why reads are being filtered out. $\endgroup$
    – Randoms
    Jul 6, 2018 at 2:17

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