How to know which sequence contains the polymorphism?

[shuang]

Below is the first polymorphic nt after pileup 2 sequences aligned to their ref genome. The file format is VCF. How do I know which sequence/file contains this polymorphism?



#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT ab3.bam ab4.bam

chr_6 40288765 . T C 79 . DP=2;AF1=1;AC1=4;DP4=0,0,2,0;MQ=60;FQ=-30.8 GT:PL:GQ 1/1:56,3,0:8 1/1:56,3,0:8

[swbarnes2]

The '1/1' in the ab3 column indicates that ab3 is predicted to be homozygous for the alternate letter there. If it said '0/1', that would mean a predicted heterozygote, '0/0' means the sample is predicted to be homozygous reference.

However, in this particular case, the QUAL score, the QC score, and the DP4 are all so poor that you can't actually trust this call. It looks like each sample has a single read at this locus.

[shuang]

Question 1.
When I pileup 3 .bam files the same polymorphic nts' 'QUAL' is much lower than when I pileup 10 .bam files. The previous 3 files are also part of the later 10 files. Why?


Question 2.
Can I process multiple (Sanger) sequences at the same time from the beginning, instead of piling them up in the end? Here is a brief summary of my protocol.

0. my data is from Sanger in .AB1 format.
1. generate fastq from ab1 via Phred and a script.
2. perform alignments via BWASW (better for read >200 bp. part of BWA package.)
3. convert from sam to bam format via Samtools.
4. pileup multiple alignment results via Samtools.