Hi!

I am baffled by lines of this VCF output by bcftools (genotype fields only shown):

GT:PL 0/0:0,15,123 0/1:28,0,82 1/1:113,12,0

~ and then a few lines later~

GT:PL 1/1:255,36,0 0/0:0,27,255 1/1:182,18,0

Note the PL entries, which are not consistent with the calls, it seems? In the upper, a high likelihood in the first PL entry has a 1/1 call, and a high value in the third PL field is called as 0/0. In the second, the opposite.

According to the v4.2 spec https://samtools.github.io/hts-specs/VCFv4.2.pdf, PL entries are ordered by the likelihood of 00,01,11, and GT entries give 0 as REF and 1,2,3,... as ALT alleles.

What have I missed?

---

MORE INFO incl. command to build vcf:

fileformat=VCFv4.2

FILTER=<ID=PASS,Description="All filters passed">

bcftoolsVersion=1.15.1+htslib-1.15.1

bcftoolsCommand=mpileup -Ou -f ../ref/GCF_000001735.4_TAIR10.1_genomic.fna

../mapping/bkground-3.final.bam ../mapping/no-muc-2.final.bam ../mapp>

reference=file://../ref/GCF_000001735.4_TAIR10.1_genomic.fna

contig=<ID=NC_003070.9,length=30427671>

contig=<ID=NC_003071.7,length=19698289>

contig=<ID=NC_003074.8,length=23459830>

contig=<ID=NC_003075.7,length=18585056>

contig=<ID=NC_003076.8,length=26975502>

contig=<ID=NC_037304.1,length=367808>

contig=<ID=NC_000932.1,length=154478>

ALT=<ID=*,Description="Represents allele(s) other than observed.">

INFO=<ID=INDEL,Number=0,Type=Flag,Description="Indicates that the variant is an INDEL.">

INFO=<ID=IDV,Number=1,Type=Integer,Description="Maximum number of raw reads supporting an indel">

INFO=<ID=IMF,Number=1,Type=Float,Description="Maximum fraction of raw reads supporting an indel">

INFO=<ID=DP,Number=1,Type=Integer,Description="Raw read depth">

INFO=<ID=VDB,Number=1,Type=Float,Description="Variant Distance Bias for filtering splice-site artefacts in RNA-seq data (bigger is

better)",Vers>

INFO=<ID=RPBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Read Position Bias (closer to 0 is better)">

INFO=<ID=MQBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Mapping Quality Bias (closer to 0 is better)">

INFO=<ID=BQBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Base Quality Bias (closer to 0 is better)">

INFO=<ID=MQSBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Mapping Quality vs Strand Bias (closer to 0 is better)">

INFO=<ID=SCBZ,Number=1,Type=Float,Description="Mann-Whitney U-z test of Soft-Clip Length Bias (closer to 0 is better)">

INFO=<ID=FS,Number=1,Type=Float,Description="Phred-scaled p-value using Fisher's exact test to detect strand bias">

INFO=<ID=SGB,Number=1,Type=Float,Description="Segregation based metric.">

INFO=<ID=MQ0F,Number=1,Type=Float,Description="Fraction of MQ0 reads (smaller is better)">

FORMAT=<ID=PL,Number=G,Type=Integer,Description="List of Phred-scaled genotype likelihoods">

FORMAT=<ID=GT,Number=1,Type=String,Description="Genotype">

INFO=<ID=AC,Number=A,Type=Integer,Description="Allele count in genotypes for each ALT allele, in the same order as listed">

INFO=<ID=AN,Number=1,Type=Integer,Description="Total number of alleles in called genotypes">

INFO=<ID=DP4,Number=4,Type=Integer,Description="Number of high-quality ref-forward , ref-reverse, alt-forward and alt-reverse

bases">

INFO=<ID=MQ,Number=1,Type=Integer,Description="Average mapping quality">

bcftools_callVersion=1.15.1+htslib-1.15.1

bcftools_callCommand=call -mv -Ov -o Athal_muc_calls.vcf; Date=Fri Jul 5 18:51:07 2024

A few sample lines of the vcf, complete:

NC_003076.8 24018928 . G A 739.009 .
DP=70;VDB=0.894089;SGB=-2.07584;MQSBZ=1.63577;FS=0;MQ0F=0;AC=6;AN=6;DP4=0,0,33,29;MQ=55 GT:PL 1/1:255,66,0 1/1:255,57,0 1/1:255,63,0 NC_003076.8
24165310 . ctttttttttttt
cTTtttttttttttt,cTTTtttttttttttt 705.096 .
INDEL;IDV=19;IMF=0.95;DP=60;VDB=0.833651;SGB=-2.0711;RPBZ=-3.42752;MQBZ=0;MQSBZ=0;BQBZ=0.303931;SCBZ=-4.88354;FS=0;MQ0F=0;AC=5,1;AN=6;DP4=0,1,32,21;MQ=60 GT:PL 1/2:255,80,59,255,0,255 1/1:255,48,0,255,34,255 1/1:255,45,0,255,30,255 NC_003076.8 24249909 . CAA
CAAA 741.951 .
INDEL;IDV=20;IMF=0.769231;DP=65;VDB=0.999029;SGB=-2.07471;RPBZ=0.532084;MQBZ=0;MQSBZ=0;SCBZ=-3.55512;FS=0;MQ0F=0;AC=6;AN=6;DP4=0,3,33,25;MQ=60 GT:PL 1/1:255,37,0 1/1:255,54,0 1/1:255,60,0 NC_003076.8
24363555 . ttatatatatatatatatatatatatatatatatatatatatatat ttatatatatatatatatatatatatatatatatatatatat 29.7787 . INDEL;IDV=1;IMF=0.5;DP=6;VDB=0.137328;SGB=-0.94647;RPBZ=-1.87867;MQBZ=0;MQSBZ=0;SCBZ=-0.707107;FS=0;MQ0F=0;AC=3;AN=4;DP4=1,0,2,0;MQ=60 GT:PL 0/1:30,33,53 1/1:60,3,0 ./.:0,0,0 NC_003076.8
24511252 . cttttttttttttt cTttttttttttttt 581.747 .
INDEL;IDV=15;IMF=0.789474;DP=55;VDB=0.0790836;SGB=-2.03626;RPBZ=-3.16804;MQBZ=0;MQSBZ=0;BQBZ=1.10185;SCBZ=0.882111;FS=0;MQ0F=0;AC=6;AN=6;DP4=1,2,15,28;MQ=60 GT:PL 1/1:240,47,9 1/1:174,22,1 1/1:220,46,19 NC_003076.8
24514611 . cttttttttttttttttttttt cTttttttttttttttttttttt 198.554 .
INDEL;IDV=10;IMF=0.625;DP=41;VDB=0.0292918;SGB=-4.50762;RPBZ=-2.30016;MQBZ=0;MQSBZ=0;BQBZ=-2.76601;SCBZ=-1.72308;FS=0;MQ0F=0;AC=4;AN=6;DP4=5,4,15,7;MQ=60 GT:PL 0/1:72,0,34 0/1:90,4,111/1:96,27,20 NC_003076.8
24648509 . caaaaaaaaaaaa cAaaaaaaaaaaaa 701.425 .
INDEL;IDV=30;IMF=0.882353;DP=83;VDB=0.0805328;SGB=-1.93982;RPBZ=-3.46456;MQBZ=-0.488678;MQSBZ=0.685119;SCBZ=-2.28166;FS=0;MQ0F=0;AC=6;AN=6;DP4=3,4,43,23;MQ=59 GT:PL 1/1:250,44,0 1/1:255,45,0 1/1:228,23,8 NC_003076.8
24817868 . t tA 741.959 .
INDEL;IDV=26;IMF=0.962963;DP=79;VDB=0.847587;SGB=-2.07802;RPBZ=0;MQBZ=0;MQSBZ=0;BQBZ=-1.84936;SCBZ=-4.9644;FS=0;MQ0F=0;AC=6;AN=6;DP4=1,2,38,35;MQ=60 GT:PL 1/1:255,67,0 1/1:255,48,0 1/1:255,75,0 NC_003076.8
24845928 . AC ACC 739.009 .
INDEL;IDV=14;IMF=0.875;DP=45;VDB=0.246007;SGB=-2.05572;RPBZ=-2.1683;MQBZ=2.82843;MQSBZ=-0.669534;BQBZ=-2.35743;SCBZ=-2.82843;FS=0;MQ0F=0;AC=6;AN=6;DP4=0,0,13,29;MQ=60 GT:PL 1/1:255,45,0 1/1:255,42,0 1/1:255,39,0 NC_003076.8
25031934 . G A 642.352 .
DP=67;VDB=0.0495212;SGB=-6.78118;RPBZ=0.62774;MQBZ=0;MQSBZ=0;BQBZ=-0.764055;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=11,8,25,21;MQ=60 GT:PL 1/1:255,51,0 0/1:169,0,189 0/1:255,0,201 NC_003076.8
25045517 . G A 656.359 .
DP=70;VDB=0.333913;SGB=-6.97044;RPBZ=0.114459;MQBZ=0;MQSBZ=0;BQBZ=0.118991;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=16,4,26,22;MQ=60 GT:PL 1/1:255,63,0 0/1:201,0,197 0/1:237,0,184 NC_003076.8
25164670 . G A 674.44 .
DP=70;VDB=0.808261;SGB=-6.21391;RPBZ=0.022777;MQBZ=0;MQSBZ=0;BQBZ=1.03132;SCBZ=-1.38013;FS=0;MQ0F=0;AC=4;AN=6;DP4=14,7,24,16;MQ=60 GT:PL 1/1:255,57,0 0/1:203,0,255 0/1:253,0,142 NC_003076.8
25171717 . atttttttttttttt aTtttttttttttttt,aTTtttttttttttttt 663.184 .
INDEL;IDV=22;IMF=0.814815;DP=69;VDB=0.0214457;SGB=-2.05574;RPBZ=-3.78464;MQBZ=0;MQSBZ=0;SCBZ=-3.45305;FS=0;MQ0F=0;AC=5,1;AN=6;DP4=3,1,30,25;MQ=60 GT:PL 1/1:225,56,14,200,19,197 1/2:255,134,136,167,0,185
1/1:255,36,0,255,66,255 NC_003076.8 25197867 .
ctttttttttt cTtttttttttt 728.488 .
INDEL;IDV=22;IMF=0.956522;DP=71;VDB=0.276288;SGB=-2.07405;RPBZ=-3.21126;MQBZ=0;MQSBZ=0;SCBZ=0;FS=0;MQ0F=0;AC=6;AN=6;DP4=3,3,27,33;MQ=60 GT:PL 1/1:255,33,0 1/1:255,63,13 1/1:255,20,0 NC_003076.8
25209132 . G A 631.268 .
DP=55;VDB=0.22647;SGB=-4.88922;RPBZ=-0.11385;MQBZ=0;MQSBZ=0;BQBZ=0.274364;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=12,10,15,11;MQ=60 GT:PL 1/1:255,33,0 0/1:164,0,255 0/1:249,0,234 NC_003076.8
25217796 . gtttttttttt gTtttttttttt 471.637 .
INDEL;IDV=13;IMF=0.722222;DP=41;VDB=0.066784;SGB=-2.0133;RPBZ=-2.58392;MQBZ=-0.492366;MQSBZ=0.899735;BQBZ=-1.14557;SCBZ=-1.27181;FS=0;MQ0F=0;AC=6;AN=6;DP4=0,2,19,15;MQ=59 GT:PL 1/1:190,28,0 1/1:183,9,01/1:122,30,0 NC_003076.8
25489380 . G A 539.441 .
DP=48;VDB=0.878792;SGB=-5.33934;RPBZ=-0.879281;MQBZ=0;MQSBZ=0;BQBZ=2.92382;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=4,11,13,18;MQ=60 GT:PL 1/1:255,57,0 0/1:170,0,161 0/1:151,0,135 NC_003076.8
25530324 . T G 724.479 .
DP=503;VDB=0.252122;SGB=-2.07944;RPBZ=0.566415;MQBZ=0;MQSBZ=0;BQBZ=-0.111853;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=35,19,233,162;MQ=60 GT:PL 0/1:255,0,93 1/1:255,60,0 0/1:255,0,54 NC_003076.8
25530330 . T G 724.455 .
DP=493;VDB=0.191416;SGB=-2.07944;RPBZ=0.849503;MQBZ=0;MQSBZ=0;BQBZ=0.314134;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=34,20,222,166;MQ=60 GT:PL 0/1:255,0,70 1/1:255,38,0 0/1:255,0,48 NC_003076.8
25531711 . C T 740.86 .
DP=577;VDB=0.569224;SGB=-2.07944;RPBZ=-0.108067;MQBZ=0;MQSBZ=0;BQBZ=1.16024;SCBZ=0.592714;FS=0;MQ0F=0;AC=6;AN=6;DP4=22,34,213,267;MQ=60 GT:PL 1/1:255,105,0 1/1:255,17,0 1/1:255,69,0 NC_003076.8
25532096 . taaaaaaaaaaa taaaaaaaaaa 621.825 .
INDEL;IDV=146;IMF=0.776596;DP=481;VDB=0.614898;SGB=-2.07944;RPBZ=-3.87462;MQBZ=0;MQSBZ=0;SCBZ=-4.66248;FS=0;MQ0F=0;AC=6;AN=6;DP4=33,34,188,187;MQ=60 GT:PL 1/1:193,85,0 1/1:210,128,0 1/1:243,156,0 NC_003076.8
25596184 . G A 675.415 .
DP=81;VDB=0.583395;SGB=-7.2535;RPBZ=0.00553127;MQBZ=0;MQSBZ=0;BQBZ=-0.373163;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=10,15,30,21;MQ=60 GT:PL 1/1:255,84,0 0/1:209,0,255 0/1:248,0,248 NC_003076.8
25616499 . G A 592.436 .
DP=68;VDB=0.344389;SGB=-6.78128;RPBZ=2.25454;MQBZ=0;MQSBZ=0;BQBZ=-1.26213;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=6,14,18,28;MQ=60 GT:PL 1/1:255,66,0 0/1:119,0,196 0/1:255,0,215 NC_003076.8
25663267 . G A 678.263 .
DP=53;VDB=0.2806;SGB=-5.55187;RPBZ=0.166885;MQBZ=0;MQSBZ=0;BQBZ=0.498906;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=12,3,19,14;MQ=60 GT:PL 1/1:255,45,0 0/1:231,0,112 0/1:229,0,189 NC_003076.8
25671509 . G A 701.36 .
DP=75;VDB=0.928342;SGB=-6.97043;RPBZ=1.82306;MQBZ=0;MQSBZ=0;BQBZ=-1.17792;SCBZ=0.629153;FS=0;MQ0F=0;AC=4;AN=6;DP4=5,14,22,26;MQ=60 GT:PL 1/1:255,66,0 0/1:255,0,236 0/1:228,0,210 NC_003076.8
25761424 . t tG 741.328 .
INDEL;IDV=23;IMF=0.766667;DP=76;VDB=0.810279;SGB=-2.07273;RPBZ=-2.42883;MQBZ=0;MQSBZ=0;SCBZ=0;FS=0;MQ0F=0;AC=6;AN=6;DP4=9,3,42,20;MQ=60 GT:PL 1/1:255,44,0 1/1:255,17,0 1/1:255,7,0 NC_003076.8
25807487 . G A 617.439 .
DP=61;VDB=0.0413344;SGB=-5.7412;RPBZ=1.63101;MQBZ=0;MQSBZ=0;BQBZ=1.98439;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=9,10,11,24;MQ=60 GT:PL 1/1:255,42,0 0/1:144,0,192 0/1:255,0,170 NC_003076.8
25863173 . G A 678.625 .
DP=55;VDB=0.893016;SGB=-6.02492;RPBZ=-1.96802;MQBZ=0;MQSBZ=0;BQBZ=0.820833;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=0,11,10,28;MQ=60 GT:PL 1/1:255,51,0 0/1:206,0,114 0/1:255,0,88 NC_003076.8
25867857 . T G 682.443 .
DP=74;VDB=0.781061;SGB=-6.59174;RPBZ=-0.115677;MQBZ=0;MQSBZ=0;BQBZ=0.579026;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=9,13,21,23;MQ=60 GT:PL 1/1:255,69,0 0/1:244,0,255 0/1:220,0,177 NC_003076.8
25909395 . catatatatatatatatatatatatatata catatatatatatatatatatatatata 57.1043 .
INDEL;IDV=2;IMF=0.666667;DP=11;VDB=0.0145983;SGB=-2.58123;RPBZ=0.103242;MQBZ=0;MQSBZ=0;SCBZ=0;FS=0;MQ0F=0;AC=3;AN=6;DP4=6,0,1,2;MQ=60 GT:PL 0/0:0,6,97 0/1:28,0,127 1/1:67,6,0 NC_003076.8
26072994 . gaaaaaaaaaaa gAaaaaaaaaaaa 484.015 .
INDEL;IDV=8;IMF=0.727273;DP=28;VDB=0.0270423;SGB=-1.93377;RPBZ=-1.77786;MQBZ=0;MQSBZ=0;BQBZ=0.168728;SCBZ=-0.890871;FS=0;MQ0F=0;AC=6;AN=6;DP4=0,0,11,13;MQ=60 GT:PL 1/1:179,24,0 1/1:168,21,0 1/1:163,24,0 NC_003076.8
26073626 . taaaaaaaaaaaaaaaaa
tAaaaaaaaaaaaaaaaaa,tAAaaaaaaaaaaaaaaaaa,tAAAAaaaaaaaaaaaaaaaaa 164.019 . INDEL;IDV=5;IMF=1;DP=12;VDB=0.323915;SGB=-1.615;MQSBZ=0;FS=0;MQ0F=0;AC=4,1,1;AN=6;DP4=0,0,8,4;MQ=60 GT:PL
1/3:119,57,45,119,57,119,69,0,69,661/2:66,45,39,26,0,23,66,45,26,66
1/1:72,12,0,72,12,72,72,12,72,72 NC_003076.8 26124485 .
G A 663.437 .
DP=63;VDB=0.7342;SGB=-6.11876;RPBZ=0.306625;MQBZ=0;MQSBZ=0;BQBZ=0.223684;SCBZ=0;FS=0;MQ0F=0;AC=4;AN=6;DP4=11,8,17,22;MQ=60 GT:PL 1/1:255,60,0 0/1:190,0,201 0/1:255,0,232 NC_003076.8
26151432 . taaaaaaaaa tAaaaaaaaaa 724.623 .
INDEL;IDV=29;IMF=0.828571;DP=90;VDB=0.187013;SGB=-2.07547;RPBZ=-3.25992;MQBZ=0;MQSBZ=0;BQBZ=-2.29504;SCBZ=0;FS=0;MQ0F=0;AC=6;AN=6;DP4=4,4,44,31;MQ=60 GT:PL 1/1:255,74,17 1/1:255,50,0 1/1:255,24,0 NC_003076.8
26200361 . ac aCc 741.865 .
INDEL;IDV=25;IMF=0.925926;DP=67;VDB=0.648932;SGB=-2.07221;RPBZ=0.952937;MQB