Hi John,

Thank you, this helps clarify things a lot. I should have read up on the
BAM/SAM format specification more before analyzing my pileup output.

Nicholas Hill

On Thu, Feb 22, 2018 at 6:29 AM, John Marshall <
john.w.marsh...@glasgow.ac.uk> wrote:

> On 21 Feb 2018, at 20:05, Nicholas Hill <nish...@ucsc.edu> wrote:
> > Say my pileup line is this:
> > chr3    73912   A       21      g,,..G,.gGGGgGGg,.Ggg
>  JJ<JJJ<sJJssJJsss7JkJ
> >
> > So at chr3:73912 the reference was an A. On the forward strand (read1),
> there are 7 guanine base pairs that aligned to the reference sequence at
> this position. Additionally, on the reverse strand (read2), there are 6
> guanine base pairs that aligned to the reference sequence at this position
> ( or is it 6 cytosine base pairs, given that it is the reverse?). This is
> where I am confused.
> It may be clearer if you mock up a small SAM file and mpileup it yourself
> so you can see what is going on.  For example,
> @SQ     SN:chr3 LN:1000000
> foo     0       chr3    73910   20      6M      *       0       0
> bar     16      chr3    73912   20      6M      *       0       0
> "samtools mpileup foobar.sam" gives  2  Gg  AB  mpileup columns at
> positions 73912-73915. The lowercase "g"s correspond to the bases of bar --
> as viewed on the reference sequence's forward strand, as one always does
> for mapped data in SAM files, as Tom noted. This read has been
> reverse-complemented (flags=16), so it was CCCCCC as viewed when it came
> off the sequencing machine, and it has been mapped to the reverse strand of
> the reference.
> > Also, what if my reference base is lowercase:
> > chr3    73912   a       21      g,,..G,.gGGGgGGg,.Ggg
>  JJ<JJJ<sJJssJJsss7JkJ
> >
> > Does this mean that the reference base is actually a thymine, given that
> it is from the reference genome?
> No, it's an A. All that mpileup documentation about upper/lower-case and
> dots/commas indicating strands pertains only to the "g,,..G,.gGGGgGGg,.Ggg"
> column.
> The reference base in column 3 is simply copied from your -f genome.fa
> file. So your genome.fa reference genome must have a mixture of upper- and
> lowercase reference bases in it. Presumably this is some kind of masking,
> but what it's supposed to indicate would be a question for whoever made
> that reference genome .fa file -- mpileup is not altering it in any way.
>     John
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