I am evaluating Mutect2 variant calling performance in GiaB mixtures (target capture, no UMI, 2000x avg coverage). In particular, I am comparing 4.0.12.0 against 4.1.9.0 with default parameters. Below, I am providing data from a representative sample.
4.1.9.0 misses variants that 4.0.12.0 was able to call. When feeding a reference VCF with option --alleles the variants are detected with decent quality scores. It is unclear why 4.1.9.0 does not make these variant calls and if this could be changed by modifying input parameters. Unlike in this issue https://github.com/broadinstitute/gatk/issues/6724 the variants were not called with the option --force-active.
These are the variants that are only called by 4.1.9.0 when the reference VCF is fed as input:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT Sample
2 25458546 . C T . . AS_SB_TABLE=723,503|25,14;DP=1302;ECNT=1;MBQ=20,20;MFRL=189,190;MMQ=60,60;MPOS=36;POPAF=7.3;TLOD=61.58 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:1226,39:0.033:1265:576,19:554,19:723,503,25,14
4 55152040 . C T . . AS_SB_TABLE=1102,1078|15,13;DP=2349;ECNT=2;MBQ=20,20;MFRL=180,164;MMQ=60,60;MPOS=35;POPAF=7.3;TLOD=31.85 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:2180,28:0.012:2208:1003,16:1104,10:1102,1078,15,13
5 170833472 . AAT A . . AS_SB_TABLE=201,501|7,17;DP=750;ECNT=1;MBQ=20,26;MFRL=203,209;MMQ=60,60;MPOS=20;POPAF=7.3;RPA=2,1;RU=AT;STR;TLOD=45.4 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:702,24:0.035:726:329,12:311,12:201,501,7,17
7 101844851 . A G . . AS_SB_TABLE=1022,1178|25,25;DP=2406;ECNT=1;MBQ=20,20;MFRL=189,198;MMQ=60,60;MPOS=46;POPAF=7.3;TLOD=65.52 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:2200,50:0.021:2250:854,29:906,19:1022,1178,25,25
7 101916798 . C A . . AS_SB_TABLE=91,916|1,37;DP=1060;ECNT=1;MBQ=32,32;MFRL=213,195;MMQ=60,60;MPOS=26;POPAF=7.3;TLOD=54.92 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:1007,38:0.033:1045:438,17:511,18:91,916,1,37
7 148506396 . A C . . AS_SB_TABLE=990,908|18,18;DP=1981;ECNT=1;MBQ=20,20;MFRL=193,203;MMQ=60,60;MPOS=35;POPAF=7.3;TLOD=48.03 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:1898,36:0.019:1934:917,15:842,17:990,908,18,18
11 108175462 . G A . . AS_SB_TABLE=682,593|10,8;DP=1354;ECNT=2;MBQ=20,20;MFRL=191,177;MMQ=60,60;MPOS=36;POPAF=7.3;TLOD=16.27 GT:AD:AF:DP:F1R2:F2R1:PGT:PID:PS:SB 1|0:1275,18:0.011:1293:597,8:622,9:1|0:108175394_T_C:108175394:682,593,10,8
12 12037318 . C G . . AS_SB_TABLE=686,588|12,7;DP=1365;ECNT=1;MBQ=20,20;MFRL=188,214;MMQ=60,60;MPOS=45;POPAF=7.3;TLOD=18.93 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:1274,19:0.014:1293:599,5:603,10:686,588,12,7
15 66679819 . G C . . AS_SB_TABLE=456,686|11,12;DP=1220;ECNT=1;MBQ=20,20;MFRL=201,184;MMQ=60,60;MPOS=49;POPAF=7.3;TLOD=30.97 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:1142,23:0.022:1165:515,15:542,8:456,686,11,12
18 42532923 . T C . . AS_SB_TABLE=951,1013|14,19;DP=2062;ECNT=1;MBQ=20,32;MFRL=183,217;MMQ=60,60;MPOS=43;POPAF=7.3;TLOD=48.91 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:1964,33:0.019:1997:909,15:910,15:951,1013,14,19
20 31019360 . AT A . . AS_SB_TABLE=1027,1027|31,25;DP=2202;ECNT=1;MBQ=20,20;MFRL=187,169;MMQ=60,60;MPOS=45;POPAF=7.3;RPA=7,6;RU=T;STR;TLOD=17.82 GT:AD:AF:DP:F1R2:F2R1:SB 0/1:2054,56:0.021:2110:965,37:987,19:1027,1027,31,25
This is how 4.0.12.0 calls these variants with standard settings:
#CHROM POS ID REF ALT QUAL FILTER INFO FORMAT Sample
2 25458546 . C T . . DP=880;ECNT=1;MBQ=36,36;MFRL=206,204;MMQ=60,60;MPOS=30;POPAF=7.3;TLOD=46.19 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:821,25:0.031:846:418,12:403,13:0.02,0.03,0.03:0.003181,0.001768,0.995
4 55152040 . C T . . DP=1582;ECNT=2;MBQ=36,36;MFRL=192,175;MMQ=60,60;MPOS=33;POPAF=7.3;TLOD=25.15 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:1425,18:0.012:1443:688,9:737,9:0.01,0.01,0.012:0.001817,0.000804,0.997
5 170833472 . AAT A . . DP=596;ECNT=1;MBQ=36,36;MFRL=211,219;MMQ=60,60;MPOS=21;POPAF=7.3;RPA=2,1;RU=AT;STR;TLOD=42.8 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:561,19:0.034:580:288,10:273,9:0.03,0.03,0.033:0.006381,0.002272,0.991
7 101844851 . A G . . DP=1820;ECNT=1;MBQ=36,36;MFRL=204,203;MMQ=60,60;MPOS=46;POPAF=7.3;TLOD=60.08 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:1630,38:0.022:1668:799,23:831,15:0.02,0.02,0.023:0.003057,0.0008676,0.996
7 101916798 . C A . . DP=1016;ECNT=1;MBQ=36,36;MFRL=212,202;MMQ=60,60;MPOS=27;POPAF=7.3;TLOD=60.48 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:964,36:0.035:1000:440,19:524,17:0.04,0.02,0.036:0.003131,0.007434,0.989
7 148506396 . A C . . DP=1399;ECNT=1;MBQ=36,36;MFRL=209,211;MMQ=60,60;MPOS=34;POPAF=7.3;TLOD=42.57 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:1327,27:0.019:1354:672,12:655,15:0.02,0.02,0.02:0.0007239,0.005058,0.994
11 108175462 . G A . . DP=972;ECNT=1;MBQ=36,36;MFRL=209,206;MMQ=60,60;MPOS=37;POPAF=7.3;TLOD=15.55 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:898,12:0.013:910:446,6:452,6:0.01,0.01,0.013:0.002966,0.0009292,0.996
12 12037318 . C G . . DP=975;ECNT=1;MBQ=36,36;MFRL=205,218;MMQ=60,60;MPOS=48;POPAF=7.3;TLOD=15.41 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:893,12:0.013:905:438,4:455,8:0.01,0.01,0.013:0.004146,0.0007942,0.995
15 66679819 . G C . . DP=870;ECNT=1;MBQ=36,36;MFRL=215,211;MMQ=60,60;MPOS=52;POPAF=7.3;TLOD=25.62 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:797,17:0.022:814:384,11:413,6:0.02,0.02,0.021:0.004622,0.001165,0.994
18 42532923 . T C . . DP=1402;ECNT=1;MBQ=36,36;MFRL=197,222;MMQ=60,60;MPOS=51;POPAF=7.3;TLOD=41.21 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:1312,25:0.019:1337:681,13:631,12:0.02,0.01,0.019:0.0009167,0.002842,0.996
20 31019360 . AT A . . DP=1530;ECNT=1;MBQ=36,36;MFRL=198,199;MMQ=60,60;MPOS=44;POPAF=7.3;RPA=7,6;RU=T;STR;TLOD=30.65 GT:AD:AF:DP:F1R2:F2R1:SAAF:SAPP 0/1:1424,34:0.022:1458:673,20:751,14:0.02,0.02,0.023:0.003063,0.0009654,0.996
Steps to reproduce
# Call without --alleles option does not produce the variants.
gatk Mutect2 --reference GRCh37.fa --read-validation-stringency LENIENT -I subset_properheader.bam -L enrichment.bed --interval-set-rule INTERSECTION -O unfiltered_noalleles.vcf.gz
# Call with --alleles option produces the variants with presumably high quality scores
gatk Mutect2 --reference GRCh37.fa --read-validation-stringency LENIENT -I subset_properheader.bam -L enrichment.bed --interval-set-rule INTERSECTION -O unfiltered_alleles.vcf.gz --alleles truth_small_variants_NA12878-NA24385-mix_sorted.vcf.gz
The BAM, BED and output VCF files are available for download here.
Bug Report
Affected tool(s) or class(es)
Mutect2
Affected version(s)
Description
I am evaluating Mutect2 variant calling performance in GiaB mixtures (target capture, no UMI, 2000x avg coverage). In particular, I am comparing 4.0.12.0 against 4.1.9.0 with default parameters. Below, I am providing data from a representative sample. 4.1.9.0 misses variants that 4.0.12.0 was able to call. When feeding a reference VCF with option
--alleles
the variants are detected with decent quality scores. It is unclear why 4.1.9.0 does not make these variant calls and if this could be changed by modifying input parameters. Unlike in this issue https://github.com/broadinstitute/gatk/issues/6724 the variants were not called with the option--force-active
.These are the variants that are only called by 4.1.9.0 when the reference VCF is fed as input:
This is how 4.0.12.0 calls these variants with standard settings:
Steps to reproduce
The BAM, BED and output VCF files are available for download here.