Issue with bam-readcount and fpfilter.pl: Saying "failed to get readcounts for variant allele"

Question

[1phamcha2]

Hi everyone,

I'm using Somatic Sniper and working on somatic mutation indel filtering using bam-readcount and fpfilter.pl, but I keep running into an issue where fpfilter.pl fails to retrieve read counts for all variants.

Below is an outline of how I processed and filtered SomaticSniper variant calls using standard filtering, bam-readcount, and fpfilter.pl:

> perl snpfilter.pl –snp-file my_sniper_file –indel-file my_indel_pileup
> perl prepare_for_readcount.pl –snp-file my_sniper_file.SNPfilter
> bam-readcount -b 15 -f my_ref.fasta -l my_sniper_file.SNPfilter.pos my_tumor.bam > my_readcounts.rc
> perl fpfilter.pl –snp-file my_sniper_file.SNPfilter –readcount-file my_readcounts.rc

However, I keep getting:
514 variants
514 failed to get readcounts for variant allele
0 had read position < 0.1
0 had strandedness < 0.005
0 had var_count < 2
0 had var_freq < 0.02
0 had mismatch qualsum difference > 50
0 had mapping quality difference > 40
0 had read length difference > 25
0 had var_distance_to_3' < 0.1
0 passed the strand filter

What I've checked so far:
-Checked that positions match in both files (my_readcounts.rc and my_sniper_file.SNPfilter)
ex. of output for readcount
chr1    759567  T   3   =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00      C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00   G:3:55.33:30.33:55.33:0:3:0.53:0.02:455.00:0:0.00:76.00:0.74        T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00 :0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    915173  C   5   =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00      C:5:45.60:29.00:45.60:5:0:0.24:0.02:425.80:5:0.74:76.00:0.74     G:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00      T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00 :0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00

ex. of output for SNPfilter
chr1    759567  .   A   G   .   .   .   GT:IGT:DP:DP4:BCOUNT:GQ:JGQ:VAQ:BQ:MQ:AMQ:SS:SSC    /0:4:0,4,0,0:4,0,0,0:39:.:0:30:60:60:0:.    1/1:3:0,0,0,3:0,0,3,0:36:.:36:30:55:55:2:44
chr1    915173  .   A   C   .   .   .   GT:IGT:DP:DP4:BCOUNT:GQ:JGQ:VAQ:BQ:MQ:AMQ:SS:SSC    /0:2:2,0,0,0:2,0,0,0:33:.:0:30:57:57:0:.    1/1:5:0,0,5,0:0,5,0,0:42:.:42:29:46:46:2:4

8

What I've tried: -Removing the normal column from the SNP file, didn't work (same results) -Adjusting the fpfilter.pl parameters so its less selective, didn't work (same results)

Any help would be greatly appreciated, thank you!