Help figuring out variant call format (vcf)

[bmtb]

Hi,

I know that there are quite a few threads already on this topic but I can't seem to find any specific to the questions I have. Anyway, I have a vcf file and can't figure out what is going on with certain lines. The file only contains biallelic SNPs and does not contain indels. Here's an example of three lines from my file:

1089 5357 S1089_5357 G A 20 PASS . GT:ADP:GQ:PL 0/0:127,0:127:99:0,255,255 0/0:1
27,10:137:99:0,52,255 0/0:43,0:43:99:0,129,255 0/1:127,19:146:99:245,0,255 0/0:127,0:127:99:0,255,255 0/
0:126,0:126:99:0,255,255 0/0:127,0:127:99:0,255,255 0/0:127,0:127:99:0,255,255 0/1:127,12:139:96:14,0,255
1089 5359 S1089_5359 - C 20 PASS . GT:ADP:GQ:PL 0/0:127,0:127:99:0,255,255 0/0:6
6,0:66:99:0,198,255 0/0:13,0:13:99:0,39,255 0/0:126,0:126:99:0,255,255 0/0:106,0:106:99:0,255,255 0/0:30,0:3
0:99:0,90,255 0/0:124,0:124:99:0,255,255 0/0:37,0:37:99:0,111,255 0/0:57,0:57:99:0,171,255
1089 5360 S1089_5360 G A 20 PASS . GT:ADP:GQ:PL 0/1:127,18:145:99:212,0,255 0/0:1
27,0:127:99:0,255,255 0/0:43,0:43:99:0,129,255 0/0:127,0:127:99:0,255,255 0/0:127,0:127:99:0,255,255 0/
0:126,0:126:99:0,255,255 0/0:127,0:127:99:0,255,255 0/0:127,0:127:99:0,255,255 0/0:127,0:127:99:0,255,255

My questions are as follows:
1) For the second line, what does the "-" mean in the column for the reference allele?
2) Should I be suspicious of SNPs that are found in close proximity to one-and-other? There are many instances in my file, such as in the above example, where numerous snps are positioned only single nts apart.
3) Lastly, many of my snps have extremely large read depths (ca. 127-254 reads per locus per sample). Should I be concerned about these snps, since they could be caused from alignment of paralogous sequences?

Any help on these questions would be great.

Thanks,
Bryan

[pallevillesen]

1) ? Reference has nothing - so it's a C insertion (?) I have never seen this in the documentation or in output... Can't help - sorry.

2) YES! That looks extremely suspicious. Check the raw alignment of the reads in IGV or similar

3) Probably mapping errors due to repeat seq or similar - remove them - You should make a histogram of the mapping depth and see if anything looks weird - but mapping errors (with mismatches) causes "SNPs/indels" to arise - and you don't want them.

My main suggestion: look at the raw data for these positions.