pileup file annotation

[NM_010117]

Hi all, I have a quick question about the pileup format. The documentation on the samtools website was clear enough, but the files that I have are slightly different.

Instead of something like this:

seq1 272 T 24  ,.$.....,,.,.,...,,,.,..^+. <<<+;<<<<<<<<<<<=<;<;7<&
seq1 273 T 23  ,.....,,.,.,...,,,.,..A <<<;<<<<<<<<<3<=<<<;<<+

I have an extra column between the reference base call, and the number of reads. The inserted column corresponds to the variant call. However, I can't actually figure out what that variant call means. At first I thought it was the automatic interpretation of the SNP corresponding to the change in amino acid, but it turns out this is not actually the case.

Here are some examples of my data:

chr1    9692883 A       R       79      79      35      54      .$,..,,,,g,,,,g..,,gg,g,g,gg,g.g..g.,.,g,g.gggg,,g.,g^Fg^F.     BaBBaRBVBHBBMBBZBBBBBBBBBBBBBaBabB^BaBBBBbBBBBBBBbBBBa
chr1    9699171 G       S       59      59      36      28      .,,,,,,,,.c,c....c....ccc,^Fc^:c        B`aL^`]YQ\FBKB]^\B`^ZaBBBBBB
chr1    9699574 A       M       75      75      35      38      ,$,,,,,,,,,cc,,,,..,.,c.,..cc..ccc^Fc^F.^F.^Fc^Fc       a\JFFFTBBBDBBBBB`\F_GE_B`aBBaaPBBBbbVB
chr1    9703423 T       K       55      55      36      39      GGGGGG.GG,.GG,..,.G,......,.........^F.^F.^:.   BBBBBBBBB`BBB]KB_BBaJGZVYK^\XT^]\a^`baa

Everything there is fairly obvious and easy to put together, except I cannot figure out what the fourth column actually corresponds to in the alignment. Obviously the SNPs are g,c,c, & G in the above example, but what do the R,S,M & K stand for?

Does anyone have any ideas?

[Chipper]

IUPAC codes (http://www.dna.affrc.go.jp/misc/MPsrch/InfoIUPAC.html).

[NM_010117]

Why the lack of confidence in the SNP call then? It seems pretty obvious from the alignment what those SNPs are. Why report it as an ambiguous nucleic acid when in actuality it's just a less well-represented polymorphism?

[swbarnes2]

Your quality scores look like they are scaled so that B is the worst quality. Which makes the quality across those positions poor.

The fact that all the alternate letters are called in the same direction, while the reference is called in both directions might not be a good sign either.

[NM_010117]

Your quality scores look like they are scaled so that B is the worst quality. Which makes the quality across those positions poor.

The fact that all the alternate letters are called in the same direction, while the reference is called in both directions might not be a good sign either.

That's a good point. I went and checked things again and it looks like the ones that are labeled confidently (i.e. as ACGT rather than with other IUPAC codes) are ones with better quality scores. Well, I'm much better off now than I was a few hours ago.

Thanks for the help.