VarScan2 - fpfilter.pl - failed to get readcounts for variant allele

Question

[vanessagpds]

Hello everyone, I'm new to variant calling and I'm having trouble running VarScan2 v2.4.4 (Support Protocol 1) which can be found in the following online document: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4278659 /

The authors advise using the fpfilter.pl accessory script (https://sourceforge.net/projects/varscan/files/scripts/). When I run the following command:

perl script_FPfilter sample.varScan.snp.filter.bed varScan.variants.readcounts –output-basename varScan.variants.filter

I get the following output:

27841 variants

27841 failed to get readcounts for variant allele

0 had read position < 0.1

0 had strandedness < 0.01

0 had var_count < 4

0 had var_freq < 0.05

0 had mismatch qualsum difference > 50

0 had mapping quality difference > 30

0 had read length difference > 25

0 had var_distance_to_3' < 0.2

0 passed the strand filter

Apparently, the problem is with obtaining the readcounts.

The bam-readcounts files were obtained as follows:

create indexed bam files

samtools index bqsr_output.bam

create bed file for sample.varScan.snp.filter

vcf2bed --snvs sample.varScan.snp.filter > sample.varScan.snp.filter.bed

run bam-read-count

bam-readcount -q 1 -b 20 -f hg38_genome.fa -l sample.varScan.snp.filter.bed bqsr_output.bam > snp_varScan.variants.readcounts

what am I doing wrong?

[ATpoint]

Can you show a head of the BED and bam-rc output?

[vanessagpds]

output Bed

chr1    15014   15015   .       .       G       C       PASS    ADP=33;WT=0;HET=1;HOM=0;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    0/1:45:33:33:20:13:39.39%:2.8029E-5:31:31:13:7:7:6
chr1    16948   16949   .       .       A       C       PASS    ADP=30;WT=0;HET=1;HOM=0;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    0/1:33:30:30:20:10:33.33%:3.9851E-4:32:31:11:9:5:5
chr1    16995   16996   .       .       T       C       PASS    ADP=34;WT=0;HET=1;HOM=0;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    0/1:53:34:34:19:15:44.12%:4.1835E-6:33:29:11:8:9:6
chr1    17967   17968   .       .       G       A       PASS    ADP=50;WT=0;HET=1;HOM=0;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    0/1:32:50:50:40:10:20%:5.9342E-4:30:33:26:14:2:8
chr1    17998   17999   .       .       C       G       PASS    ADP=29;WT=0;HET=1;HOM=0;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    0/1:19:29:29:23:6:20.69%:1.1736E-2:27:29:13:10:3:3
chr1    24785   24786   .       .       G       A       PASS    ADP=22;WT=0;HET=1;HOM=0;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    0/1:35:22:22:12:10:45.45%:2.6061E-4:28:33:6:6:6:4
chr1    187484  187485  .       .       G       A       PASS    ADP=36;WT=0;HET=0;HOM=1;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    1/1:139:36:36:6:30:83.33%:1.1855E-14:27:33:4:2:15:15
chr1    187496  187497  .       .       G       A       PASS    ADP=43;WT=0;HET=1;HOM=0;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    0/1:47:43:43:29:14:32.56%:1.727E-5:27:33:16:13:6:8
chr1    268515  268516  .       .       C       T       PASS    ADP=39;WT=0;HET=1;HOM=0;NC=0    GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    0/1:118:39:39:11:28:71.79%:1.3724E-12:30:32:4:7:9:19
chr1    629905  629906  .       .       C       T       PASS    ADP=122;WT=0;HET=0;HOM=1;NC=0   GT:GQ:SDP:DP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR    1/1:255:122:122:3:119:97.54%:2.2028E-67:33:32:1:2:35:84

output bam-readcount

chr1    15014   T       34      =: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:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  T:34:2.47:32.21:2.47:20:14:0.59:0.00:11.97:20:0.20:65.94:0.22   N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    15015   G       33      =: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:13:2.54:31.31:2.54:7:6:0.57:0.00:31.31:7:0.20:63.85:0.23   G:20:2.40:31.35:2.40:13:7:0.58:0.00:0.00:13:0.19:68.35:0.21     T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    16948   A       30      =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:30:35.23:33.23:35.23:16:14:0.34:0.00:28.73:16:0.48:121.67:0.32        C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00       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  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    16949   A       30      =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:20:22.85:32.90:22.85:11:9:0.40:0.00:13.20:11:0.27:121.90:0.26 C:10:60.00:31.30:60.00:5:5:0.23:0.00:59.80:5:0.90:121.20:0.43        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  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    16995   A       34      =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:34:42.82:32.56:42.82:20:14:0.71:0.00:25.94:20:0.62:108.91:0.39        C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00       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  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    16996   T       34      =: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:15:60.00:29.00:60.00:9:6:0.90:0.00:49.87:9:0.63:113.33:0.43        G:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  T:19:29.26:33.68:29.26:11:8:0.57:0.00:7.05:11:0.61:105.42:0.35  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    17967   G       50      =: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:50:1.44:31.26:1.44:28:22:0.63:0.00:16.90:28:0.35:92.62:0.33   T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    17968   G       50      =:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  A:10:1.00:33.00:1.00:2:8:0.68:0.00:36.10:2:0.26:85.20:0.36      C:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00       G:40:1.55:30.98:1.55:26:14:0.60:0.00:12.10:26:0.35:94.47:0.32   T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    17998   C       28      =: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:25:1.88:30.04:1.88:14:11:0.60:0.00:12.68:14:0.31:109.48:0.39       G:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  T:3:1.00:31.67:1.00:2:1:0.22:0.00:31.67:2:0.05:93.67:0.22       N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00
chr1    17999   C       29      =: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:23:1.96:27.91:1.96:13:10:0.61:0.00:11.35:13:0.32:110.17:0.39       G:6:1.00:29.00:1.00:3:3:0.24:0.00:30.00:3:0.05:95.83:0.31       T:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00  N:0:0.00:0.00:0.00:0:0:0.00:0.00:0.00:0:0.00:0.00:0.00