Real SNPs discovered with samtools v0.1.7 disappear with v0.1.8

[geraldes]

Dear all,

(Sorry I posted this earlier in the genome sequencing forum, nut Bioinformatics might be a better forum for it)...

I used sanger sequencing to confirm some SNPs that I "discovered" with a GAII experiment using samtools v0.1.7. For a "bunch" of reasons, I generated a new pileup file using v0.1.8. The pileup looks like this for v0.1.7:

scaffold_7 13782966 A R 155 155 49 354 G$G$G$G$G$.$g$g$g$G,,.g,...G...GGG.,,.G,,,GG,g,g....G..G.,G..Ggggggg.Gg,ggg.GGG.G.,gg...g,.Ggg.,gG.,,,gG....g..GGGG.,,..ggg.G.GGg,.G.GG.gGGG..GGG,.,g,...GG,g
.G,g..,ggG.G...g.,,,.gggG.,,,.GGgg,,,GGG....G,GGG.GG,.G.GG.,..G...ggG,.GG..gg..Gg,,GG.G..GG....G.,.,GgGGG.G.Gg,G.G,,,,,.GGG.G.G.G,GGG.GGggg,g,.GGG.GG,G,,,gg.G...g.,ggG....G,gg,.g,,GG.GG.G.g,^].^]G^]G^]G^]G^]G^].^F.^].^F.^F.^]g^].^],^]G^F
, .-....787-77.87-.........-78..787..7777----...--7....777787..78877......-777...87-.77-78-.7877...-.7.......773377733332873222337778778878777777777*7777777787787777777777777777877878787%7778777727778773777777277777733837737833877-..
7777777777-787.8.7-7778878..777.....7777888777.788777..-...8777878.7..-..77777.7..-777887.-..7...77778877-.88778888877-7.7. FF]]]FF]FFFFF]]FF]]]]]]FFFF]FF]]]]]FFFFFF]FFF]]FFF]]]FFF]]F]F]]]]FFFF]]FFFFFF]]]F]FFFF]]F]F]FFFFFFF]FFF]]FF]
]F]F]]]]]]F]F]]]]FF]F]]FF]]F]]]]FFFF]]]]]]F]FFF]FF]]]FF]FF]]F]]FF]]]FF]]]]]FFFFF]]]]FFF]]]FFFFFFFF]]FFFFFFF]]]]F]]]]]]]FF]FFFFFF]]]FFF]]F]]]]FFFF]]]]F]]FF]]F:FFFFF]]]]FFFF]]]]FFFF]FF]]]F]]]FFFFFFF]FFFF]]]]]FFF]]FF]]F]F]FFFFF]]FFF]]]]]]]]

And like this for v.0.1.8:
scaffold_7 13782966 A A 1 0 49 354 G$G$G$G$G$.$g$g$g$G,,.g,...G...GGG.,,.G,,,GG,g,g....G..G.,G..Ggggggg.Gg,ggg.GGG.G.,gg...g,.Ggg.,gG.,,,gG....g..GGGG.,,..ggg.G.GGg,.G.GG.gGGG..GGG,.,g,...GG,g
.G,g..,ggG.G...g.,,,.gggG.,,,.GGgg,,,GGG....G,GGG.GG,.G.GG.,..G...ggG,.GG..gg..Gg,,GG.G..GG....G.,.,GgGGG.G.Gg,G.G,,,,,.GGG.G.G.G,GGG.GGggg,g,.GGG.GG,G,,,gg.G...g.,ggG....G,gg,.g,,GG.GG.G.g,^].^]G^]G^]G^]G^]G^].^F.^].^F.^F.^]g^].^],^]G^F
, .-....787-77.87-.........-78..787..7777----...--7....777787..78877......-777...87-.77-78-.7877...-.7.......773377733332873222337778778878777777777*7777777787787777777777777777877878787%7778777727778773777777277777733837737833877-..
7777777777-787.8.7-7778878..777.....7777888777.788777..-...8777878.7..-..77777.7..-777887.-..7...77778877-.88778888877-7.7. FF]]]FF]FFFFF]]FF]]]]]]FFFF]FF]]]]]FFFFFF]FFF]]FFF]]]FFF]]F]F]]]]FFFF]]FFFFFF]]]F]FFFF]]F]F]FFFFFFF]FFF]]FF]
]F]F]]]]]]F]F]]]]FF]F]]FF]]F]]]]FFFF]]]]]]F]FFF]FF]]]FF]FF]]F]]FF]]]FF]]]]]FFFFF]]]]FFF]]]FFFFFFFF]]FFFFFFF]]]]F]]]]]]]FF]FFFFFF]]]FFF]]F]]]]FFFF]]]]F]]FF]]F:FFFFF]]]]FFFF]]]]FFFF]FF]]]F]]]FFFFFFF]FFFF]]]]]FFF]]FF]]F]F]FFFFF]]FFF]]]]]]]]
F]FF]]]]F

How can the consensus quality and SNP quality go from 155 to 0 and 1?

Thanks

[Jon_Keats]

First suggestion would be to try the current version 0.12a to make sure this is not a bug that was fixed a year ago.

[swbarnes2]

Try rerunning the pileup or mpileup command with -B. This will disable the new BAQ calculations. That might be why your SNPs are being downgraded.

[Jon_Keats]

Something is odd with your phred scales, were these multiple runs? Were they all converted to sanger phred+33 scale? The values look to go from the middle of sanger phred+33 to the top of illumin phred+64 values, so something is wrong.

[geraldes]

Thanks for the comments. These are very helpful.

Yes, I will be trying to run mpileup with version 0.13.
As far as the phred scales go. No, these were not multiple runs. The problem is that you can't see where the base qualities ("7" "8" "." and "-") end and the mapping qualities ("F" and "]") begin...