Samtools SNP calling

[vidhya]

Hello there
I have a issue with sam SNP calling. I work with captured genomic sequences.
The fold coverage is very high at 600X. I used BWA (mismatch penalty -7) to map the reads to the genome and used samtools to call SNPs. I used mpileup and then realised that a known SNP was not called by mpileup and I tried to investigate what is happening in that region with pileup. The output is as follows

CFA15 1299612 T T 255 0 59 309 g,GG,,.g,,.G.g.,,,gg.G,gg,g,,.G.,,,,,,GG...gGG,,,g.ggggg,.ggG.gg..g,Gg.Gggg.G
.g,,.gg,,,,gg.,g,g.,,.Gg,.,gg,gg,ggggG,..Ggg,,g,g,,,.g,gG,gGgg.g,GG,Gg,,..,,g.G,,,,,,,.G,,gg,gG.gg,gGGGg.GGGG,g,gGg,G,,g,,g,.,g,g..GG.G.,gggg
GggG.G,,g,g,.,,..,gGG..G,G,,..g,gg,g,.,Ggg,.G,g,.,gGGGGg,G.GGg,.gggG,g,,,g,G.G..G...,g^]g^],^],^]g^]G !T!!]^^!^^^!^!^^^^!!^!^!!^!^^^!^^^^^^
^!!^^^!!!^^^!^!!!!!^^!!!^!!^^!^!!^!!!!^!^!^^^!!^^^^!!^^!^!^^^^!!^^^!!^!!^!!!!!^^^!!!^^!^!^^^^!^!!^!!!!^!^!!^!!^^^^^^!^!^^^^^^^^!^^!!^!!^!!^!!
!!!^!!!!^!^!!!^!^^!^^!^^^!^!^^!!^!^^!!!!!!!!^!^^!]!^^^^^^^!!!^^!^!^^^B!^!!^!^^^!!!^^!^!^^^!!!!,!^!^!!!^^!!!!^!^__!_!_!EE!EEA>!!EE!!

What I do not understand is why is samtools not reporting the consensus sequence as K ? Is this the reason why it is not called as variant position ?
Thanks a lot for the answers

[swbarnes2]

All those !'s are the lowest quality. It doesn't want to call a G because it thinks all the G calls are horribly unreliable.

Did you run mpileup with the default settings? At my fingertips, I've got a similar case, with 2 SNPs that definitely sanger confirmed, but also had lousy quality scores. When I re-ran mpileup with -B, the quality scores improved to what the .sam file said they ought to be, and my two SNPs popped up.

If you ran pileup, you should really get the newest version of samtools, and run mpileup. People will be less willing to troubleshoot software they know is superseded.

[kissthefuture]

do you have format the original data?If not ,I also understand...

[vidhya]

Thank you very much. It worked.