What is " ./.:. " in the GT output of VCF/BCF

Question

[rightmirem]

I have 70 BAM files (1 for each of 70 complete genome samples), against which I ran mpileup and bcftools call to get 70 BCF variant files. I then merged all these BCF files into one large BCF file (using bcftools merge). The question is how to interpret lines (like the following).

For example, this line appears to say - amongst all 70 samples - there is ONE sample which had both ALT alleles (1/1:54,3,0). The AC=2 and the AN=2...so I assumed that the multiple ./.:. meant no matching data (or, implicitely, that all the other samples matched the reference).

1       10297   .       C       T       27.3033 PASS    VDB=0.96;SGB=-0.453602;RPB=1;MQB=1;BQB=1;MQ0F=0.166667;MQ=40;DP=6;DP4=0,1,0,2;AN=2;AC=2 GT:PL   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:. 1/1:54,3,0   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.   ./.:.

HOWEVER, to check this I ran ...

ls ./*bcf | while read filename; do bcftools view -H -i 'POS=10297'  $filename ; done

...which searched all 70 of the individual sample BCF files for data at position 10297. I got 56 lines back.1 matching the 1/1:54,3,0 ...

1   10297   .   C   T   27.3033 PASS    DP=6;VDB=0.96;SGB=-0.453602;RPB=1;MQB=1;BQB=1;MQ0F=0.166667;AC=2;AN=2;DP4=0,1,0,2;MQ=40 GT:PL   1/1:54,3,0

(see the rest of the output below)

I notice also that 55 of the returns are annotated GL000208, which I assume is a reference to this contig. The only return that does NOT reference GL000208 is the one that was heterozygous 1/1 (the rest here homozygous 0/1).

So, I realize I probably just answered my own question (that GL000208 is a known SNP?? in the reference...or perhaps just IS the reference??)...but confirmation and maybe some details would be helpful :D

=== THE OTHER 55 RETURNS ===

GL000208.1  10297   .   T   A   222 PASS    DP=81;VDB=0.0460824;SGB=-0.693145;RPB=0.487662;MQB=1;MQSB=1;BQB=0.407941;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=23,12,20,21;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=72;VDB=0.562051;SGB=-0.69312;RPB=0.998414;MQB=1;MQSB=1;BQB=0.873005;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=18,12,20,12;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=98;VDB=0.208973;SGB=-0.693145;RPB=0.702471;MQB=1;MQSB=1;BQB=0.541047;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=22,28,21,19;MQ=60    GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   110 PASS    DP=61;VDB=0.0147504;SGB=-0.670168;RPB=0.21281;MQB=1;MQSB=1;BQB=0.434287;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=23,21,6,4;MQ=60  GT:PL0/1:145,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=81;VDB=0.592406;SGB=-0.692717;RPB=0.418206;MQB=1;MQSB=1;BQB=0.129543;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=25,30,13,10;MQ=60    GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=68;VDB=0.0306362;SGB=-0.693079;RPB=0.505598;MQB=1;MQSB=1;BQB=0.309834;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=20,16,16,13;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=90;VDB=0.889266;SGB=-0.69312;RPB=0.967791;MQB=1;MQSB=1;BQB=0.372578;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=29,27,17,15;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=98;VDB=0.0613164;SGB=-0.693079;RPB=0.999491;MQB=1;MQSB=1;BQB=0.000509874;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=31,36,14,15;MQ=60    GT:PL   0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=120;VDB=0.159068;SGB=-0.693147;RPB=0.929583;MQB=1;MQSB=1;BQB=0.063241;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=43,23,21,31;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=88;VDB=0.457547;SGB=-0.69311;RPB=0.985837;MQB=1;MQSB=1;BQB=0.133031;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=24,30,17,14;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=80;VDB=0.0588969;SGB=-0.693145;RPB=0.940626;MQB=1;MQSB=1;BQB=0.80705;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=22,16,22,18;MQ=60    GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   118 PASS    DP=13;VDB=0.136193;SGB=-0.616816;RPB=0.194278;MQB=0.961166;MQSB=0.961166;BQB=0.531709;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=5,2,2,4;MQ=6GT:PL  0/1:151,0,156

GL000208.1  10297   .   T   A   222 PASS    DP=88;VDB=0.825491;SGB=-0.692976;RPB=0.591208;MQB=1;MQSB=1;BQB=0.00869542;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=34,25,12,14;MQ=60  GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=105;VDB=0.322342;SGB=-0.693146;RPB=0.837101;MQB=1;MQSB=1;BQB=0.0363126;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=29,30,23,20;MQ=60  GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=29;VDB=0.00495249;SGB=-0.690438;RPB=0.43845;MQB=1;MQSB=1;BQB=0.163206;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=8,4,9,8;MQ=60   GT:PL0/1:255,0,233

GL000208.1  10297   .   T   A   222 PASS    DP=75;VDB=0.0750914;SGB=-0.693079;RPB=0.915284;MQB=1;MQSB=1;BQB=0.0100281;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=22,23,13,16;MQ=60  GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   212 PASS    DP=73;VDB=0.0565414;SGB=-0.69168;RPB=0.889463;MQB=1;MQSB=1;BQB=0.00821477;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=25,29,10,9;MQ=60   GT:PL0/1:246,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=101;VDB=0.0915851;SGB=-0.693097;RPB=0.999383;MQB=1;MQSB=1;BQB=0.0638933;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=33,34,14,16;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=121;VDB=0.0283351;SGB=-0.693146;RPB=0.941197;MQB=1;MQSB=1;BQB=0.00317092;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=39,32,26,18;MQ=60    GT:PL   0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=68;VDB=0.654808;SGB=-0.692914;RPB=0.276665;MQB=1;MQSB=1;BQB=0.49648;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=24,15,15,10;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=77;VDB=0.157913;SGB=-0.692352;RPB=0.968799;MQB=1;MQSB=1;BQB=0.225269;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=31,22,12,9;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=110;VDB=0.219827;SGB=-0.693139;RPB=0.777912;MQB=1;MQSB=1;BQB=0.316287;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=39,34,15,21;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=125;VDB=0.367966;SGB=-0.693147;RPB=0.666486;MQB=1;MQSB=1;BQB=0.106854;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=40,30,24,28;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=133;VDB=0.926022;SGB=-0.693147;RPB=0.313402;MQB=1;MQSB=1;BQB=0.688419;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=34,28,38,25;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=95;VDB=0.122979;SGB=-0.693147;RPB=0.863786;MQB=1;MQSB=1;BQB=0.334625;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=21,24,19,30;MQ=60    GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   219 PASS    DP=72;VDB=0.633915;SGB=-0.692067;RPB=0.911418;MQB=1;MQSB=1;BQB=0.972641;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=23,25,9,11;MQ=60 GT:PL0/1:252,0,255

GL000208.1  10297   .   T   A   217 PASS    DP=88;VDB=0.849872;SGB=-0.692914;RPB=0.859387;MQB=1;MQSB=1;BQB=0.77073;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=28,35,17,8;MQ=60  GT:PL0/1:250,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=110;VDB=0.889949;SGB=-0.693147;RPB=0.833134;MQB=1;MQSB=1;BQB=0.0618411;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=26,25,30,28;MQ=60  GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=114;VDB=0.0551714;SGB=-0.693147;RPB=0.992354;MQB=1;MQSB=1;BQB=0.192874;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=34,23,25,29;MQ=60  GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=80;VDB=0.49346;SGB=-0.692352;RPB=0.835525;MQB=1;MQSB=1;BQB=0.645775;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=28,25,13,8;MQ=60  GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=102;VDB=0.182212;SGB=-0.693079;RPB=0.946461;MQB=1;MQSB=1;BQB=0.283315;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=38,26,12,17;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=92;VDB=0.779094;SGB=-0.69311;RPB=0.904512;MQB=1;MQSB=1;BQB=0.948066;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=27,33,16,15;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=95;VDB=0.318542;SGB=-0.693127;RPB=0.927494;MQB=1;MQSB=1;BQB=0.959119;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=31,27,20,13;MQ=60    GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=74;VDB=0.356374;SGB=-0.692831;RPB=0.575503;MQB=1;MQSB=1;BQB=0.63342;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=24,24,15,9;MQ=60  GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=62;VDB=0.0243716;SGB=-0.692717;RPB=0.187277;MQB=1;MQSB=1;BQB=0.996656;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=20,18,13,10;MQ=60   GT:PL0/1:255,0,255

1   10297   .   C   T   27.3033 PASS    DP=6;VDB=0.96;SGB=-0.453602;RPB=1;MQB=1;BQB=1;MQ0F=0.166667;AC=2;AN=2;DP4=0,1,0,2;MQ=40 GT:PL   1/1:54,3,0

GL000208.1  10297   .   T   A   222 PASS    DP=85;VDB=0.112395;SGB=-0.693144;RPB=0.970041;MQB=1;MQSB=1;BQB=0.256217;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=15,30,20,19;MQ=60    GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=94;VDB=0.142519;SGB=-0.693147;RPB=0.998392;MQB=1;MQSB=1;BQB=0.764039;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=26,20,23,25;MQ=60    GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=113;VDB=0.212235;SGB=-0.693147;RPB=0.979879;MQB=1;MQSB=1;BQB=0.212548;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=27,34,23,29;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=123;VDB=0.478991;SGB=-0.693147;RPB=0.903964;MQB=1;MQSB=1;BQB=0.177772;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=40,34,23,24;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   187 PASS    DP=54;VDB=0.10893;SGB=-0.686358;RPB=0.428026;MQB=1;MQSB=1;BQB=0.315058;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=21,19,6,8;MQ=60   GT:PL0/1:221,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=62;VDB=0.40128;SGB=-0.692717;RPB=0.299916;MQB=1;MQSB=1;BQB=0.306744;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=17,22,11,12;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=49;VDB=0.651545;SGB=-0.69168;RPB=0.325662;MQB=1;MQSB=1;BQB=0.944296;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=17,13,10,9;MQ=60  GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=102;VDB=0.0519816;SGB=-0.693145;RPB=0.992323;MQB=1;MQSB=1;BQB=0.0857568;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=24,32,24,17;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   219 PASS    DP=55;VDB=0.540591;SGB=-0.689466;RPB=0.852376;MQB=1;MQSB=1;BQB=0.0104413;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=23,16,7,9;MQ=60 GT:PL0/1:252,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=69;VDB=0.0665008;SGB=-0.692914;RPB=0.704969;MQB=1;MQSB=1;BQB=0.395949;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=22,20,13,12;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=107;VDB=0.832778;SGB=-0.693147;RPB=0.637084;MQB=1;MQSB=1;BQB=0.178158;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=30,25,28,22;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=61;VDB=0.222952;SGB=-0.692067;RPB=0.139278;MQB=1;MQSB=1;BQB=0.291918;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=23,16,12,8;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   205 PASS    DP=70;VDB=0.163372;SGB=-0.691153;RPB=0.674838;MQB=1;MQSB=1;BQB=0.0225;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=28,24,11,7;MQ=60   GT:PL0/1:239,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=148;VDB=0.00266433;SGB=-0.693147;RPB=0.896319;MQB=1;MQSB=1;BQB=0.00888503;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=47,24,41,34;MQ=60   GT:PL   0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=87;VDB=0.577098;SGB=-0.69311;RPB=0.999613;MQB=1;MQSB=1;BQB=0.133484;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=28,25,16,15;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   222 PASS    DP=97;VDB=0.249946;SGB=-0.693147;RPB=0.359953;MQB=1;MQSB=1;BQB=0.993045;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=15,25,31,21;MQ=60    GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   221 PASS    DP=95;VDB=0.46429;SGB=-0.692717;RPB=0.826527;MQB=1;MQSB=1;BQB=0.859249;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=36,29,12,11;MQ=60 GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   221 PASS    DP=109;VDB=0.848602;SGB=-0.692914;RPB=0.478419;MQB=1;MQSB=1;BQB=0.986804;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=41,32,15,10;MQ=60   GT:PL0/1:255,0,255

GL000208.1  10297   .   T   A   26.9567 PASS    DP=59;VDB=0.0175011;SGB=-0.636426;RPB=0.109536;MQB=1;MQSB=1;BQB=0.689018;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=22,28,4,3;MQ=60 GT:PL0/1:63,0,255

GL000208.1  10297   .   T   A   166 PASS    DP=56;VDB=0.0376046;SGB=-0.686358;RPB=0.991632;MQB=1;MQSB=1;BQB=0.237299;MQ0F=0;ICB=1;HOB=0.5;AC=1;AN=2;DP4=20,16,12,2;MQ=60    GT:PL0/1:200,0,255

Answer 1

[finswimmer]

Hello,

the first column in the vcf describe the chromosom on which the variant is located. "1" and "GL000208.1" are not the same. "1" is chromosom 1 and "GL000208.1" seems to be an alternate scaffold that couldn't be mapped to a chromosome.

So the merging result is correct. You just have in one sample the variant on chromosome 1 at position 10297. The other that looks similiar were mapped to GL000208.1.

fin swimmer

Answer 2

[Medhat]

./. means that there is not enough information. for sample to match the reference ( homozygous reference) it need to be 0/0

from documentation

If a call cannot be made for a sample at a given locus, ‘.’ should be specified for each missing allele in the GT field (for example ‘ ./. ’ for a diploid genotype and ‘.’ for haploid genotype). The meanings of the separators are as follows (see the PS field below for more details on incorporating phasing information into the genotypes):

• / : genotype unphased
• | : genotype phased

and that is depend on the threshold you used to call varaint

The PL

PL : the phred-scaled genotype likelihoods rounded to the closest integer (and otherwise defined precisely as the GL field) (Integers)

Answer 3

[rightmirem]

Actually, to add more confusion...

Contig GL000208 at position 10297 is a "T"...

10201 gaagatattt ccttttccac catagccctc aaagtgctcc aaatgtccac ttgcagattc
10261 tacgttaaga gtgtttcaaa actgctctgt caaaagTtag gttaaccctg tgagttgaat
10321 gcacacatca caaagtactt tttgagaatg cttctgtcta gtttttaggt gaagatattc

...which does appear to align with all the other 55 lines - meaning that they are all 0/1 @ 10297 T->A

However the one 1/1match says 1 10297 . C T. Doesn't this mean REF=C but ALT(1)=T ?

Very confused :)

[Medhat]

I would advice you to add this to the question because it is not an answer to your question.