VQSR PASS "Variants" with AC=0, AF=0, is this normal behavior?

I am using GATK 3.4-0 / VQSR to evaluate variants called in ~300 germline genomes of varying coverage (8x-40x). When I do so, many "variants" pass using even a stringent tranche, despite not having a QUAL value or having AC=0 / AF=0. Additionally, some PASS variants have data fields GT:AD:DP, and others have GT:AD:DP:GQ:PL. Why does this occur?

For instance, VQSR Tranche SNP 99.00 to 99.90: -4.4822 <= x < -0.1962

Example:
chr21 1419 . T G . PASS AC=0;AF=0.00;AN=524;BaseQRankSum=-1.733e+00;DP=5194;MQ=31.17;MQ0=0;MQRankSum=0.00;NCC=1;ReadPosRankSum=1.73;SNPEFF_EFFECT=INTERGENIC;SNPEFF_FUNCTIONAL_CLASS=NONE;SNPEFF_IMPACT=MODIFIER;VQSLOD=1.67;culprit=DP GT:AD:DP 0/0:2,0:2 0/0:14,0:14 0/0:25,0:25 0/0:1,0:1 0/0:20,0:20 0/0:36,0:36 ./.:8,2 0/0:4,0:4 0/0:4,0:4 0/0:12,0:12 0/0:38,0:38 0/0:3,0:3 0/0:39,0:39 0/0:39,0:39 0/0:14,0:14 0/0:22,0:22 0/0:4,0:4 0/0:22,0:22 0/0:17,0:17 0/0:25,0:25 0/0:32,0:32 0/0:3,0:3 0/0:32,0:32 0/0:30,0:30 0/0:7,0:7 0/0:76,0:76 0/0:28,0:28 0/0:14,0:14 0/0:36,0:36 0/0:24,0:24 0/0:24,0:24 0/0:17,0:17 0/0:7,0:7 0/0:37,0:37 0/0:23,0:23 0/0:23,0:23 0/0:20,0:20 0/0:4,0:4 0/0:4,0:4 0/0:31,0:31 0/0:3,0:3 0/0:24,0:24 0/0:9,0:9 0/0:13,0:13 0/0:5,0:5 0/0:38,0:38 0/0:5,0:5 0/0:23,0:23 0/0:16,0:16 0/0:11,0:11 0/0:32,0:32 0/0:38,0:38 0/0:56,0:56 0/0:28,0:28 0/0:32,0:32 0/0:46,0:46 0/0:18,0:18 0/0:6,0:6 0/0:29,0:29 0/0:7,0:7 0/0:50,0:50 0/0:14,0:14 0/0:33,0:33 0/0:19,0:19 0/0:33,0:33 0/0:28,0:28 0/0:26,0:26 0/0:6,0:6 0/0:19,0:19 0/0:19,0:19 0/0:26,0:26 0/0:23,0:23 0/0:16,0:16 0/0:26,0:26 0/0:22,0:22 0/0:20,0:20 0/0:18,0:18 0/0:18,0:18 0/0:29,0:29 0/0:1,0:1 0/0:34,0:34 0/0:67,0:67 0/0:22,0:22 0/0:16,0:16 0/0:37,0:37 0/0:19,0:19 0/0:5,0:5 0/0:23,0:23 0/0:26,0:26 0/0:24,0:24 0/0:31,0:31 0/0:27,0:27 0/0:23,0:23 0/0:33,0:33 0/0:6,0:6 0/0:42,0:42 0/0:9,0:9 0/0:7,0:7 0/0:34,0:34 0/0:16,0:16 0/0:28,0:28 0/0:5,0:5 0/0:11,0:11 0/0:25,0:25 0/0:39,0:39 0/0:22,1:23 0/0:28,0:28 0/0:15,0:15 0/0:30,0:30 0/0:19,0:19 0/0:5,0:5 0/0:2,0:2 0/0:2,0:2 0/0:8,0:8 0/0:3,0:3 0/0:16,0:16 0/0:31,0:31 0/0:5,0:5 0/0:26,0:26 0/0:20,0:20 0/0:14,0:14 0/0:23,0:23 0/0:49,0:49 0/0:14,0:14 0/0:23,0:23 0/0:48,0:48 0/0:52,1:53 0/0:5,0:5 0/0:6,0:6 0/0:31,0:31 0/0:44,0:44 0/0:7,0:7 0/0:10,0:10 0/0:18,0:18 0/0:14,1:15 0/0:11,0:11 0/0:16,0:16 0/0:17,0:17 0/0:22,0:22 0/0:24,1:25 0/0:15,0:15 0/0:21,0:21 0/0:11,0:11 0/0:12,0:12 0/0:5,1:6 0/0:51,0:51 0/0:40,0:40 0/0:63,0:63 0/0:49,0:49 0/0:73,0:73 0/0:90,0:90 0/0:38,0:38 0/0:56,0:56 0/0:43,0:43 0/0:39,0:39 0/0:45,0:45 0/0:41,0:41 0/0:58,0:58 0/0:22,0:22 0/0:25,0:25 0/0:28,0:28 0/0:30,0:30 0/0:17,0:17 0/0:5,0:5 0/0:2,0:2 0/0:6,0:6 0/0:6,0:6 0/0:16,0:16 0/0:10,0:10 0/0:4,0:4 0/0:11,0:11 0/0:5,0:5 0/0:3,0:3 0/0:2,0:2 0/0:2,0:2 0/0:10,0:10 0/0:22,0:22 0/0:15,0:15 0/0:13,0:13 0/0:22,0:22 0/0:10,0:10 0/0:17,0:17 0/0:15,0:15 0/0:14,0:14 0/0:33,0:33 0/0:14,0:14 0/0:22,0:22 0/0:21,0:21 0/0:22,1:23 0/0:20,0:20 0/0:18,0:18 0/0:14,0:14 0/0:29,0:29 0/0:12,0:12 0/0:21,0:21 0/0:13,0:13 0/0:23,0:23 0/0:21,0:21 0/0:20,0:20 0/0:13,0:13 0/0:9,0:9 0/0:13,0:13 0/0:14,0:14 0/0:23,0:23 0/0:19,0:19 0/0:14,0:14 0/0:19,0:19 0/0:22,0:22 0/0:10,0:10 0/0:15,0:15 0/0:26,0:26 0/0:30,0:30 0/0:16,0:16 0/0:9,0:9 0/0:12,0:12 0/0:12,0:12 0/0:11,0:11 0/0:17,0:17 0/0:10,0:10 0/0:12,0:12 0/0:16,0:16 0/0:9,0:9 0/0:9,0:9 0/0:11,0:11 0/0:14,0:14 0/0:3,0:3 0/0:12,0:12 0/0:5,0:5 0/0:4,0:4 0/0:9,0:9 0/0:4,0:4 0/0:19,0:19 0/0:21,0:21 0/0:6,0:6 0/0:7,0:7 0/0:10,0:10 0/0:13,0:13 0/0:11,0:11 0/0:2,0:2 0/0:9,0:9 0/0:9,0:9 0/0:5,0:5 0/0:6,0:6 0/0:32,0:32 0/0:7,0:7 0/0:12,0:12 0/0:8,0:8 0/0:2,0:2 0/0:19,0:19 0/0:9,0:9 0/0:3,0:3 0/0:8,0:8 0/0:19,0:19 0/0:7,0:7 0/0:9,0:9 0/0:14,0:14 0/0:7,0:7 0/0:8,0:8 0/0:5,0:5 0/0:11,0:11 0/0:14,0:14 0/0:8,1:9 0/0:12,0:12

chr21 1432 . C T 1609.66 PASS AC=5;AF=9.542e-03;AN=524;BaseQRankSum=3.28;DP=6194;FS=0.000;GQ_MEAN=60.76;GQ_STDDEV=44.35;InbreedingCoeff=-0.0114;MLEAC=5;MLEAF=9.542e-03;MQ=39.30;MQ0=0;MQRankSum=0.780;NCC=1;QD=13.64;ReadPosRankSum=-5.450e-01;SNPEFF_EFFECT=INTERGENIC;SNPEFF_FUNCTIONAL_CLASS=NONE;SNPEFF_IMPACT=MODIFIER;SOR=0.627;VQSLOD=0.834;culprit=FS GT:AD:DP:GQ:PL 0/0:2,0:2:6:0,6,49 0/0:35,0:35:99:0,102,1530 0/0:39,0:39:93:0,93,1395 0/0:7,0:7:18:0,18,270 0/0:24,0:24:72:0,72,637 0/0:40,0:40:99:0,108,1620 0/0:2,0:2:6:0,6,47 0/0:7,0:7:21:0,21,184 0/0:6,0:6:18:0,18,160 0/0:17,1:18:23:0,23,376 0/0:24,0:24:66:0,66,990 0/0:9,0:9:21:0,21,315 0/0:51,0:51:99:0,120,1800 0/0:39,0:39:99:0,108,1620 0/0:9,0:9:27:0,27,240 0/0:18,0:18:48:0,48,720 0/0:7,0:7:18:0,18,270 0/0:24,0:24:69:0,69,1035 0/0:31,0:31:78:0,78,1170 0/1:18,18:.:99:492,0,409 0/0:30,0:30:84:0,84,1260 0/0:16,0:16:48:0,48,437 0/0:25,0:25:66:0,66,990 0/0:27,0:27:63:0,63,945 0/0:11,0:11:33:0,33,284 0/0:82,0:82:99:0,120,1800 0/0:63,0:63:99:0,120,1800 0/1:2,7:.:19:188,0,19 0/0:45,0:45:99:0,120,1800 0/0:35,0:35:90:0,90,1350 0/0:47,0:47:99:0,120,1800 0/0:16,0:16:48:0,48,451 0/0:19,0:19:45:0,45,675 0/0:47,0:47:99:0,120,1800 0/0:30,0:30:72:0,72,1080 0/0:22,0:22:54:0,54,810 0/0:7,0:7:18:0,18,270 0/0:2,0:2:6:0,6,63 0/0:5,0:5:12:0,12,180 0/0:66,0:66:99:0,120,1800 0/0:6,0:6:15:0,15,225 0/0:28,0:28:75:0,75,1125 0/0:13,1:14:11:0,11,313 0/0:12,0:12:33:0,33,495 0/0:16,0:16:45:0,45,675 0/0:58,0:58:99:0,120,1800 0/0:24,0:24:66:0,66,990 0/0:27,0:27:78:0,78,1170 0/0:25,0:25:66:0,66,990 0/0:25,0:25:75:0,75,660 0/0:41,0:41:99:0,114,1710 0/0:24,0:24:60:0,60,900 0/0:48,0:48:99:0,120,1800 0/0:44,0:44:99:0,120,1800 0/0:30,0:30:81:0,81,1215 0/0:48,0:48:99:0,120,1800 0/0:23,0:23:69:0,69,605 0/0:3,0:3:9:0,9,75 0/0:33,0:33:84:0,84,1260 0/0:8,0:8:21:0,21,315 0/0:75,0:75:99:0,120,1800 0/0:31,0:31:87:0,87,1305 0/0:39,0:39:99:0,111,1665 0/0:15,1:16:20:0,20,356 0/0:61,0:61:99:0,120,1800 0/0:28,0:28:75:0,75,1125 0/0:38,0:38:99:0,102,1530 0/0:11,0:11:30:0,30,450 0/0:15,0:15:45:0,45,449 0/0:24,0:24:72:0,72,633 0/0:29,0:29:84:0,84,1260 0/0:27,0:27:72:0,72,1080 0/0:19,0:19:54:0,54,810 0/0:24,0:24:72:0,72,634 0/0:21,0:21:57:0,57,855 0/0:18,0:18:51:0,51,765 0/0:14,0:14:39:0,39,585 0/0:20,0:20:48:0,48,720 0/0:27,0:27:72:0,72,1080 0/0:2,0:2:3:0,3,45 0/0:32,0:32:81:0,81,1215 0/0:65,0:65:99:0,120,1800 0/0:22,0:22:66:0,66,581 0/0:15,0:15:39:0,39,585 0/0:56,0:56:99:0,120,1800 0/1:13,17:.:99:474,0,273 0/0:8,0:8:21:0,21,315 0/0:23,1:24:60:0,60,580 0/0:29,0:29:75:0,75,1125 0/0:36,0:36:96:0,96,1440 0/0:31,0:31:90:0,90,1350 0/0:29,0:29:78:0,78,1170 0/0:26,0:26:69:0,69,1035 0/0:33,0:33:87:0,87,1305 0/0:13,0:13:30:0,30,450 0/0:64,0:64:99:0,120,1800 0/0:8,0:8:24:0,24,201 0/0:12,0:12:36:0,36,342 0/0:38,0:38:93:0,93,1395 0/0:13,0:13:30:0,30,450 0/0:38,0:38:99:0,105,1575 0/0:6,0:6:15:0,15,225 0/0:6,0:6:18:0,18,141 0/0:26,0:26:75:0,75,1125 0/0:63,0:63:99:0,120,1800 0/0:9,0:9:24:0,24,360 0/0:22,0:22:63:0,63,945 0/0:17,0:17:45:0,45,675 0/0:54,0:54:99:0,120,1800 0/0:18,0:18:45:0,45,675 0/0:9,0:9:24:0,24,360 0/0:2,0:2:6:0,6,63 0/0:5,0:5:12:0,12,180 0/0:11,0:11:30:0,30,450 0/0:4,0:4:9:0,9,135 0/0:11,0:11:30:0,30,450 0/0:33,0:33:87:0,87,1305 0/0:4,0:4:12:0,12,100 0/0:23,0:23:60:0,60,900 0/0:29,0:29:78:0,78,1170 0/0:18,0:18:45:0,45,675 0/1:14,15:.:99:392,0,276 0/0:65,0:65:99:0,120,1800 0/0:31,0:31:78:0,78,1170 0/0:44,0:44:99:0,117,1755 0/0:93,0:93:99:0,120,1800 0/0:75,0:75:99:0,120,1800 0/0:6,0:6:18:0,18,155 0/0:7,0:7:15:0,15,225 0/0:33,0:33:93:0,93,1395 0/0:45,0:45:99:0,120,1800 0/0:17,0:17:45:0,45,675 0/0:14,0:14:42:0,42,377 0/0:28,0:28:78:0,78,1170 0/0:14,0:14:42:0,42,392 0/0:15,0:15:45:0,45,384 0/0:14,0:14:39:0,39,585 0/0:24,0:24:66:0,66,99 0/0:18,0:18:51:0,51,765 0/0:17,0:17:42:0,42,630 0/0:24,0:24:63:0,63,945 0/0:25,0:25:72:0,72,1080 0/0:16,0:16:48:0,48,413 0/0:14,0:14:36:0,36,54 0/0:6,0:6:15:0,15,225 0/0:56,0:56:99:0,120,1800 0/0:47,1:48:99:0,120,1800 0/0:68,0:68:99:0,120,1800 0/0:62,1:63:99:0,120,1800 0/0:64,0:64:99:0,120,1800 0/0:108,1:109:99:0,120,1800 0/0:60,2:62:99:0,120,1800 0/0:76,0:76:99:0,120,1800 0/0:38,0:38:99:0,108,1620 0/0:45,0:45:99:0,120,1800 0/0:39,1:40:87:0,87,1009 0/0:48,0:48:99:0,120,1800 0/0:33,0:33:90:0,90,1350 0/0:29,0:29:75:0,75,1125 0/0:33,0:33:81:0,81,1215 0/0:29,0:29:87:0,87,759 0/0:28,0:28:69:0,69,1035 0/0:22,0:22:60:0,60,900 0/0:7,0:7:21:0,21,183 0/0:6,0:6:15:0,15,225 0/0:7,0:7:15:0,15,225 0/0:15,0:15:36:0,36,540 0/0:9,1:10:4:0,4,195 0/0:7,0:7:21:0,21,175 0/0:11,0:11:21:0,21,315 0/0:4,0:4:9:0,9,135 0/0:9,0:9:27:0,27,239 0/0:5,0:5:12:0,12,180 0/0:7,0:7:21:0,21,197 0/0:7,0:7:18:0,18,270 0/0:13,0:13:30:0,30,450 0/0:18,0:18:51:0,51,765 0/0:18,0:18:51:0,51,765 0/0:17,0:17:51:0,51,442 0/0:21,0:21:54:0,54,810 0/0:19,0:19:51:0,51,765 0/0:22,0:22:57:0,57,855 0/0:16,0:16:45:0,45,675 0/0:22,0:22:66:0,66,594 0/0:30,0:30:75:0,75,1125 0/0:23,0:23:63:0,63,945 0/0:8,0:8:24:0,24,237 0/0:21,0:21:57:0,57,855 0/0:14,0:14:42:0,42,389 0/0:14,0:14:39:0,39,585 0/0:13,0:13:36:0,36,540 0/0:15,0:15:45:0,45,371 0/0:11,0:11:33:0,33,266 0/0:24,0:24:69:0,69,1035 0/0:21,0:21:57:0,57,855 0/0:16,0:16:42:0,42,630 0/0:22,0:22:66:0,66,579 0/0:19,0:19:54:0,54,810 0/0:19,0:19:54:0,54,810 0/0:22,0:22:63:0,63,945 0/0:17,0:17:45:0,45,675 0/0:20,0:20:60:0,60,534 0/0:12,0:12:36:0,36,323 0/0:16,0:16:42:0,42,630 0/0:18,0:18:45:0,45,675 0/0:20,0:20:54:0,54,810 0/0:23,0:23:63:0,63,945 0/0:7,0:7:21:0,21,188 0/0:18,0:18:51:0,51,765 0/0:9,0:9:27:0,27,241 0/0:16,0:16:48:0,48,439 0/0:21,0:21:51:0,51,765 0/0:13,0:13:30:0,30,450 0/0:11,0:11:33:0,33,301 0/0:15,0:15:42:0,42,630 0/0:5,0:5:12:0,12,180 0/0:10,0:10:27:0,27,405 0/0:22,0:22:57:0,57,855 0/0:10,0:10:30:0,30,268 0/0:9,0:9:21:0,21,315 0/0:18,0:18:42:0,42,630 0/0:9,0:9:24:0,24,360 0/0:10,0:10:27:0,27,405 0/0:12,0:12:33:0,33,495 0/0:21,0:21:57:0,57,855 0/0:7,0:7:18:0,18,270 0/0:23,0:23:60:0,60,900 0/0:16,0:16:48:0,48,433 0/0:18,0:18:48:0,48,720 0/0:9,0:9:27:0,27,241 0/0:11,0:11:33:0,33,269 0/0:22,0:22:57:0,57,855 0/0:22,0:22:60:0,60,900 0/1:7,7:.:99:185,0,123 0/0:15,0:15:42:0,42,630 0/0:16,0:16:39:0,39,585 0/0:21,0:21:54:0,54,810 0/0:13,0:13:36:0,36,540 0/0:14,0:14:39:0,39,585 0/0:14,0:14:42:0,42,380 0/0:12,0:12:33:0,33,495 0/0:8,0:8:24:0,24,199 0/0:10,0:10:27:0,27,405 0/0:36,0:36:93:0,93,1395 0/0:8,0:8:24:0,24,170 0/0:10,0:10:30:0,30,248 0/0:13,0:13:39:0,39,305 ./.:0,0:0 0/0:24,0:24:69:0,69,1035 0/0:17,0:17:42:0,42,630 0/0:12,0:12:36:0,36,298 0/0:32,0:32:90:0,90,1350 0/0:22,0:22:66:0,66,535 0/0:9,0:9:27:0,27,216 0/0:17,0:17:42:0,42,630 0/0:17,0:17:39:0,39,585 0/0:11,0:11:30:0,30,450 0/0:18,0:18:54:0,54,417 0/0:11,0:11:27:0,27,405 0/0:17,1:18:40:0,40,404 0/0:22,0:22:63:0,63,945 0/0:6,0:6:18:0,18,162 0/0:14,1:15:34:0,34,351

Answers

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @agaricx
    Hi,

    Can you tell me a little more about how you processed your data? Like, did you follow the GATK Best Practices? What tools did you run on your data (from the beginning) and what kind of samples are you working with?

    Thanks,
    Sheila

  • Hi Sheila. Thank you for your reply.
    The data was assembled with BWA, sorted with SAMtools, marked with Picard, and IndelRealigner / BQSR of GATK... following best practices. Variants were called individually with HaplotypeCaller 3.3-0 in gVCF mode, then joint called before proceeding to VQSR in SNP mode, followed by INDEL mode. Subsequently annotated with SNPEff.

    They are nonhuman samples with a good annotated genome, a SNP chip and dbSNP dataset used for training,

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @agaricx
    Hi,

    Okay. Can you post the exact commands you ran in the VQSR step?

    Thanks,
    Sheila

  • Sure thing!

    java -Xmx48g -jar GenomeAnalysisTK.jar -T VariantRecalibrator -R genome.fa -input INPUT.vcf.gz -nt 12 \
    -resource:hdchip,known=false,training=true,truth=true,prior=15.0 SNPchip.vcf \
    -resource:dbsnp,known=true,training=false,truth=false,prior=4.0 dbSNP.vcf \
    -an DP -an QD -an FS -an SOR -mode SNP \
    -tranche 100.0 -tranche 99.9 -tranche 99.0 -tranche 90.0 \
    -recalFile SNP_recal.output.recal \
    -tranchesFile SNP_recal.output.tranches \
    -rscriptFile SNP_recal.output.plots.R;
    java -Xmx48g -jar GenomeAnalysisTK.jar -R genome.fa -T ApplyRecalibration -input INPUT.vcf.gz --ts_filter_level 99.0 -tranchesFile SNP_recal.output.tranches -recalFile SNP_recal.output.recal -mode SNP -o 990.SNP.vcf.gz;

    java -Xmx48g -jar GenomeAnalysisTK.jar -T VariantRecalibrator -R genome.fa -input 990.SNP.vcf.gz -nt 12 \
    -resource:indel2015,known=true,training=true,truth=true,prior=6.0 indel2015.vcf \
    -an DP -an QD -an MQRankSum -an SOR -an ReadPosRankSum -an FS -mode INDEL \
    -tranche 100.0 -tranche 99.9 -tranche 99.0 -tranche 90.0 \
    -recalFile INDEL_recal.output.recal \
    -tranchesFile INDEL_recal.output.tranches \
    -rscriptFile INDEL_recal.output.plots.R;
    java -Xmx48g -jar GenomeAnalysisTK.jar -R $genome.fa -T ApplyRecalibration -input 990.SNP.vcf.gz --ts_filter_level 99.0 -tranchesFile INDEL_recal.output.tranches -recalFile INDEL_recal.output.recal -mode INDEL -o 990.FINAL.vcf.gz;

    Any help is greatly appreciated.

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @agaricx
    Hi,

    I just went back to your original question and realized you are asking why with a stringent tranche, many variants are passing. The issue is that you are confusing tranche meaning! I am sorry for not catching this earlier. Tranche level 90 is the most stringent, while 100 is the most lenient. Basically, tranche level 90 it means you are keeping only the top 90% of variants. But, tranche level 100 means you are keeping essentially all the variants. Have a look at the last paragraph in this article: https://www.broadinstitute.org/gatk/guide/article?id=2805

    -Sheila

  • Hi Sheila,
    Thank you, but I understand what a tranche is, and in this case I simply gave the example of a 99.0 tranche. I don't use tranches for variant pruning, and use VQSLOD filters instead. The issue is that I have variants that PASS even at 90.0 or have acceptable VQSLOD, but are not variant at all (AC=0, AF=0, homozygous for reference).

    If it matters, I call bases at every position before joint calling using GenotypeGVCFs:
    GenomeAnalysisTK.jar -T HaplotypeCaller --emitRefConfidence BP_RESOLUTION --variant_index_type LINEAR -A Coverage -A AlleleBalance -A HaplotypeScore -A BaseCounts -A ReadPosRankSumTest -A QualByDepth --output_mode EMIT_ALL_SITES

  • SheilaSheila Broad InstituteMember, Broadie, Moderator

    @agaricx
    Hi,

    Oh dear. I thought I had solved the problem! :smile:
    Okay. Can you post the plots and tranches file?

    Thanks,
    Sheila

  • tommycarstensentommycarstensen United KingdomMember

    @agaricx Is AC=1 before or after annotation with SNPEff? Did you run GenotypeGVCFs after HaplotypeCaller? It might be beneficial to post your commands?

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