QC for indels - Githubissues

gevro commented 10 months ago

Hi, Do you have recommended steps for evaluating general quality of indel calls for each sample? For example for single base substitutions there are the mismatch QC files. Anything analogous for indels?

We have some samples for which we suspect a higher background of false positive indels (we use standard indel filter settings), and we also saw that in Abascal et al. there were a few samples excluded from indel analysis, but we're not sure which QC evals would be best.

Thanks!

gevro commented 10 months ago

Just to add, I noticed some indels marked as PASS that have DP4 values indicating that alt reads were only found on the fwd but not the rev strand. I would assume for duplex calling, the indels should be observed in both fwd and rev reads? For example:

chr2    33731063    .   T   TTA 221.417 PASS    INDEL;IDV=10;IMF=1;DP=10;VDB=6.99472e-07;SGB=-0.670168;FS=0;MQ0F=0;AC=1;AN=1;DP4=0,0,0,10;MQ=60;RB=chr2,33730903,33731088,TTT,TAT;SEQ=CCATATCATTATATATAT    GT:PL:DP:DV:SP:DP4  1:251,0:10:10:0:0,0,0,10

Not sure if this is an additional filter we should be looking at, or perhaps any other INFO or FORMAT tags.

fa8sanger commented 10 months ago

Hi,

Regarding your first question, the samples with high rates of indels in our paper were contaminated samples. I managed to filter germline SNPs from the contaminant but not indels, hence we didn’t do indel analyses for those samples. Have a look into verifybamid contamination estimates.

Regarding your second question, forward and reverse refer to the mapping orientation, and that doesn’t correspond with the two original DNA strands. Indeed, both strands will map with the same orientation, one in f1r2 and the other in f2r1. Have a look at Extended data fig 1 to better understand this.

On 26 Dec 2023, at 19:41, gevro @.***> wrote:

Just to add, I noticed some indels marked as PASS that have DP4 values indicating that alt reads were only found on the fwd but not the rev strand. I would assume for duplex calling, the indels should be observed in both fwd and rev reads? For example:

chr2 33731063 . T TTA 221.417 PASS INDEL;IDV=10;IMF=1;DP=10;VDB=6.99472e-07;SGB=-0.670168;FS=0;MQ0F=0;AC=1;AN=1;DP4=0,0,0,10;MQ=60;RB=chr2,33730903,33731088,TTT,TAT;SEQ=CCATATCATTATATATAT GT:PL:DP:DV:SP:DP4 1:251,0:10:10:0:0,0,0,10

Not sure if this is an additional filter we should be looking at, or perhaps any other INFO or FORMAT tags.

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gevro commented 10 months ago

Thanks, understood. Any other QC filters useful for indels, or is FILTER = PASS the only required filter?

fa8sanger commented 10 months ago

That’s the only one we use

On 26 Dec 2023, at 20:45, gevro @.***> wrote:

Thanks, understood. Any other QC filters useful for indels, or is FILTER = PASS the only required filter?

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gevro commented 10 months ago

Just FYI - we identified the source of most of the indel false positives. The new NovaseqX instrument has a different sequencing error pattern than Novaseq 6000, so when somatic and germline reference samples are sequenced on different instruments, it can cause this. There are not many loci like this, and they are easy to spot because these false positives are recurrent across multiple independent samples. So we will just add these to the NOISE mask. We are sure these are not contamination because the artifact pattern matches the instrument discordance, not the library prep batch, and this only seems to cause recurrent indel artifacts, not SNP artifacts.

fa8sanger commented 10 months ago

That’s very interesting, thanks for letting us know. I’ve noticed problems with NovaseqX too, but they affected substitutions, not indels. There were many recurrent artefacts in extended sequence contexts, seen in many samples. We are planning to add those to the NOISE mask as well but I haven’t had a chance yet. Working with a NovaSeqX matched normal alleviates the problem but doesn’t solve it. I believe Illumina is investigating this. I’d love to know more about the indel artefacts. Do they happen in specific sequence contexts?

On 27 Dec 2023, at 23:55, gevro @.***> wrote:

Just FYI - we identified the source of most of the indel false positives. The new NovaseqX instrument has a different sequencing error pattern than Novaseq 6000, so when somatic and germline reference samples are sequenced on different instruments, it can cause this. There are not many loci like this, and they are easy to spot because these false positives are recurrent across multiple independent samples. So we will just add these to the NOISE mask. We are sure these are not contamination because the artifact pattern matches the instrument discordance, not the library prep batch, and this only seems to cause recurrent indel artifacts, not SNP artifacts.

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gevro commented 10 months ago

Interesting. We didn't see recurrent artifacts in SNPs. How did you identify these, in case we didn't do the search properly?

Regarding indels: upon further analysis, it seems that most of the Novaseq X indel recurrent artifacts we found happen even when the germline reference was also sequenced on Novaseq X. We also found one region with recurrent artifacts for Novaseq samples.

Here are recurrent indels we found in >=5 samples in two different datasets of ~30 independent samples each. One dataset was only Novaseq 6000, and one dataset was mostly Novaseq X.

First column is # of samples in which the indel was detected

Dataset 1 (only Novaseq 6000)
28      chr3    93470660        .       GC      G
19      chr3    93470714        .       TTGA    T
15      chr3    93470568        .       G       GA
7       chr3    93470677        .       AT      A
7       chr3    93470535        .       GT      G
6       chr3    93470719        .       AT      A
6       chr3    93470592        .       TA      T
6       chr3    93470407        .       AT      A
6       chr1    193234361       .       A       AAATTAT
5       chr3    93470681        .       T       TC
5       chr3    93470664        .       TG      T
5       chr3    93470631        .       T       TCA
5       chr3    93470543        .       AC      A
5       chr2    192297664       .       T       TCCAAAGATA

Dataset 2 (mostly NovaseqX)

22      chr3    93470691        .       AT      A
12      chr18   110665  .       TC      T
10      chr5    6039630 .       G       GTTGT
10      chr5    49599504        .       GA      G
10      chr2    80565600        .       G       GAAAC
9       chr9    121816555       .       C       CGT
9       chr22   47573548        .       G       GTGTA
9       chr22   37390607        .       A       AG
8       chr3    93470681        .       T       TC
8       chr20   15637220        .       G       GTATTA
7       chr5    79157401        .       G       GT
7       chr5    24244570        .       G       GT
7       chr5    117097233       .       G       GATGT
7       chr4    21851143        .       T       TGC
7       chr3    93470660        .       GC      G
7       chr2    235849481       .       T       TCTC
7       chr18   37633970        .       T       TGACCTTG
7       chr13   108112528       .       A       AGATTAC
7       chr12   4848250 .       T       TTG
7       chr1    218078822       .       G       GT
6       chr7    155919569       .       A       AG
6       chr6    92310257        .       G       GA
6       chr4    143285483       .       A       ACTC
6       chr21   37727458        .       C       CGT
6       chr2    129201728       .       A       ATATG
6       chr12   108979090       .       T       TATATATATGC
6       chr11   42534858        .       G       GGT
6       chr10   46545261        .       A       AT
5       chrX    139912749       .       T       TTTTTA
5       chr9    116409870       .       G       GCA
5       chr7    9172174 .       A       AT
5       chr7    20447188        .       C       CCT
5       chr7    153920212       .       G       GAGC
5       chr6    96110820        .       TA      T
5       chr6    79565540        .       T       TATC
5       chr3    6183442 .       AT      A
5       chr3    26930008        .       C       CTTCCT
5       chr3    198112059       .       T       TCTGC
5       chr3    113073671       .       A       AT
5       chr2    20592148        .       TG      T
5       chr2    182194985       .       G       GC
5       chr20   31189252        .       G       GACTCA
5       chr18   72842070        .       A       AT
5       chr17   71534023        .       T       TAC
5       chr14   63259340        .       A       AC
5       chr13   70014560        .       T       TAAGA
5       chr1    16727339        .       A       AC
5       chr11   43195703        .       C       CAT
5       chr11   128162675       .       C       CGTGTGTGT

You can see one indel artifact region that is in both types of Novaseq -- a chr3 SATELLITE artifact region (chr3:93470362-93470799). I'm assuming you have seen this too. It is not in the standard hg38 NOISE mask.

But you can see for Novaseq X, there is a lager diversity of regions with recurrent indels. We don't see a clear pattern why these regions are doing this.

The issue is that without figuring out the pattern, we don't know how many of the non-recurrent indels are artifacts. Perhaps there are other artifacts hiding among the indels found in only one sample.

Anyway, appreciate hearing what you all figure out about this.

fa8sanger commented 10 months ago

Thank you very much for sending this. I’ll look into this when I am back at work next week. We’ve been doing our analyses with GRCh37, I wonder if that could explain the difference.

Which version of the pipeline are you using? There was a bug in the indel calling pipeline in one of the versions.

These are some examples of recurrent substitution artefacts (coordinates in GRCh37). I grabbed the extended sequence context and did a multiple sequence alignment, revealing a quite specific extended context.

1 86182030 . A G . PASSBTAG=TTA|GCA;BBEG=86181983;BEND=86182369;TRI=ATC>C;QPOS=47;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=1;DEPTH_NORM_REV=18;DPLX_ASXS=106;DPLX_CLIP=0;DPLX_NM=3.5;BULK_ASXS=124;BULK_NM=0 5 10205136 . T C . PASSBTAG=AAC|TAC;BBEG=10204954;BEND=10205166;TRI=ATC>C;QPOS=30;DEPTH_FWD=5;DEPTH_REV=5;DEPTH_NORM_FWD=14;DEPTH_NORM_REV=15;DPLX_ASXS=102;DPLX_CLIP=0;DPLX_NM=1.2;BULK_ASXS=90;BULK_NM=0 7 117464982 . A G . PASSBTAG=TGG|CAC;BBEG=117464939;BEND=117465167;TRI=ATC>C;QPOS=43;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=20;DPLX_ASXS=110;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=119;BULK_NM=1 8 52086040 . T C . PASSBTAG=CCC|TCT;BBEG=52085854;BEND=52086080;TRI=ATC>C;QPOS=40;DEPTH_FWD=5;DEPTH_REV=10;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=14;DPLX_ASXS=115;DPLX_CLIP=0;DPLX_NM=1.7;BULK_ASXS=119;BULK_NM=1 9 86349715 . A G . PASSBTAG=CCA|CCT;BBEG=86349674;BEND=86349916;TRI=ATC>C;QPOS=41;DEPTH_FWD=6;DEPTH_REV=4;DEPTH_NORM_FWD=4;DEPTH_NORM_REV=20;DPLX_ASXS=106;DPLX_CLIP=0;DPLX_NM=1.5;BULK_ASXS=113;BULK_NM=1 11 18214938 . T C . PASSBTAG=CCC|TGT;BBEG=18214779;BEND=18215025;TRI=ATC>C;QPOS=87;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=14;DEPTH_NORM_REV=2;DPLX_ASXS=114;DPLX_CLIP=0;DPLX_NM=1.8;BULK_ASXS=114;BULK_NM=0 16 80731751 . T C . PASSBTAG=CGT|TTG;BBEG=80731509;BEND=80731789;TRI=ATC>C;QPOS=38;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=4;DPLX_ASXS=87;DPLX_CLIP=0;DPLX_NM=2.8;BULK_ASXS=96;BULK_NM=1 19 24266074 . T C . PASSBTAG=GCC|TGA;BBEG=24265837;BEND=24266093;TRI=ATC>C;QPOS=19;DEPTH_FWD=8;DEPTH_REV=7;DEPTH_NORM_FWD=23;DEPTH_NORM_REV=2;DPLX_ASXS=107;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=102;BULK_NM=0 X 94152579 . A G . PASSBTAG=TCT|CGG;BBEG=94152512;BEND=94152792;TRI=ATC>C;QPOS=67;DEPTH_FWD=6;DEPTH_REV=4;DEPTH_NORM_FWD=9;DEPTH_NORM_REV=15;DPLX_ASXS=97;DPLX_CLIP=0;DPLX_NM=3.5;BULK_ASXS=86;BULK_NM=1 3 34027554 . A G . PASSBTAG=ATT|TGA;BBEG=34027527;BEND=34027813;TRI=ATC>C;QPOS=27;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=5;DEPTH_NORM_REV=17;DPLX_ASXS=84;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=72;BULK_NM=1 3 167192858 . T C . PASSBTAG=TTT|CTT;BBEG=167192595;BEND=167192869;TRI=ATC>C;QPOS=11;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=6;DPLX_ASXS=119;DPLX_CLIP=0;DPLX_NM=1;BULK_ASXS=125;BULK_NM=1 7 79332957 . T C . PASSBTAG=AGG|GTT;BBEG=79332556;BEND=79332997;TRI=ATC>C;QPOS=40;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=21;DEPTH_NORM_REV=4;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=3.5;BULK_ASXS=97;BULK_NM=1 7 113215431 . T C . PASSBTAG=ATC|GAA;BBEG=113215217;BEND=113215465;TRI=ATC>C;QPOS=34;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=8;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=2.3;BULK_ASXS=97;BULK_NM=0 11 18214938 . T C . PASSBTAG=CTG|TCC;BBEG=18214736;BEND=18214971;TRI=ATC>C;QPOS=33;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=9;DEPTH_NORM_REV=7;DPLX_ASXS=117;DPLX_CLIP=0;DPLX_NM=1.5;BULK_ASXS=127;BULK_NM=1 19 24266074 . T C . PASSBTAG=TGA|CGA;BBEG=24265863;BEND=24266091;TRI=ATC>C;QPOS=17;DEPTH_FWD=5;DEPTH_REV=2;DEPTH_NORM_FWD=28;DEPTH_NORM_REV=4;DPLX_ASXS=103;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=97;BULK_NM=1 4 30299975 . T C . PASSBTAG=GCG|GTG;BBEG=30299770;BEND=30300082;TRI=ATC>C;QPOS=107;DEPTH_FWD=5;DEPTH_REV=4;DEPTH_NORM_FWD=26;DEPTH_NORM_REV=2;DPLX_ASXS=108;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1 6 86953825 . T C . PASSBTAG=TCC|TAT;BBEG=86953604;BEND=86953844;TRI=ATC>C;QPOS=19;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=8;DEPTH_NORM_REV=15;DPLX_ASXS=116;DPLX_CLIP=0;DPLX_NM=1;BULK_ASXS=127;BULK_NM=0 7 102604434 . T C . PASSBTAG=GCT|ACG;BBEG=102604074;BEND=102604480;TRI=ATC>C;QPOS=46;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=9;DPLX_ASXS=90;DPLX_CLIP=0;DPLX_NM=2.3;BULK_ASXS=101;BULK_NM=0 19 10702606 . A G . PASSBTAG=TCA|CTA;BBEG=10702584;BEND=10702842;TRI=ATC>C;QPOS=22;DEPTH_FWD=5;DEPTH_REV=6;DEPTH_NORM_FWD=6;DEPTH_NORM_REV=12;DPLX_ASXS=74;DPLX_CLIP=0;DPLX_NM=3.7;BULK_ASXS=88;BULK_NM=3 6 138492211 . A G . PASSBTAG=GTA|AGC;BBEG=138492140;BEND=138492399;TRI=ATC>C;QPOS=71;DEPTH_FWD=3;DEPTH_REV=3;DEPTH_NORM_FWD=7;DEPTH_NORM_REV=18;DPLX_ASXS=85;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=111;BULK_NM=0 7 37443388 . A G . PASSBTAG=AGA|CGA;BBEG=37443365;BEND=37443561;TRI=ATC>C;QPOS=23;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=14;DEPTH_NORM_REV=15;DPLX_ASXS=82;DPLX_CLIP=0;DPLX_NM=1.5;BULK_ASXS=87;BULK_NM=1 8 34728286 . T C . PASSBTAG=TTT|AGG;BBEG=34728045;BEND=34728309;TRI=ATC>C;QPOS=23;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=23;DEPTH_NORM_REV=10;DPLX_ASXS=102;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=103;BULK_NM=0 11 18214938 . T C . PASSBTAG=AAA|AAA;BBEG=18214656;BEND=18214974;TRI=ATC>C;QPOS=36;DEPTH_FWD=5;DEPTH_REV=3;DEPTH_NORM_FWD=9;DEPTH_NORM_REV=7;DPLX_ASXS=116;DPLX_CLIP=0;DPLX_NM=1.6;BULK_ASXS=127;BULK_NM=1 11 113861932 . T C . PASSBTAG=CTC|ATC;BBEG=113861576;BEND=113861954;TRI=ATC>C;QPOS=22;DEPTH_FWD=3;DEPTH_REV=3;DEPTH_NORM_FWD=24;DEPTH_NORM_REV=4;DPLX_ASXS=118;DPLX_CLIP=0;DPLX_NM=1.3;BULK_ASXS=129;BULK_NM=0 3 168095318 . A G . PASSBTAG=CTT|ACC;BBEG=168095250;BEND=168095540;TRI=ATC>C;QPOS=68;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=8;DEPTH_NORM_REV=17;DPLX_ASXS=68;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=89;BULK_NM=1 18 43314618 . T C . PASSBTAG=AGG|CCC;BBEG=43314377;BEND=43314634;TRI=ATC>C;QPOS=16;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=14;DPLX_ASXS=99;DPLX_CLIP=0;DPLX_NM=3.7;BULK_ASXS=103;BULK_NM=1 4 143523440 . T C . PASSBTAG=GGT|TCT;BBEG=143523223;BEND=143523567;TRI=ATC>C;QPOS=127;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=6;DPLX_ASXS=84;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=79;BULK_NM=0

On 29 Dec 2023, at 15:55, gevro @.***> wrote:

Interesting. We didn't see recurrent artifacts in SNPs. How did you identify these, in case we didn't do the search properly?

Regarding indels: upon further analysis, it seems that most of the Novaseq X indel recurrent artifacts we found happen even when the germline reference was also sequenced on Novaseq X. We also found one region with recurrent artifacts for Novaseq samples.

Here are recurrent indels we found in >=5 samples in two different datasets of ~30 independent samples each. One dataset was only Novaseq 6000, and one dataset was mostly Novaseq X.

First column is # of samples in which the indel was detected

Dataset 1 (mostly Novaseq 6000) 28 chr3 93470660 . GC G 19 chr3 93470714 . TTGA T 15 chr3 93470568 . G GA 7 chr3 93470677 . AT A 7 chr3 93470535 . GT G 6 chr3 93470719 . AT A 6 chr3 93470592 . TA T 6 chr3 93470407 . AT A 6 chr1 193234361 . A AAATTAT 5 chr3 93470681 . T TC 5 chr3 93470664 . TG T 5 chr3 93470631 . T TCA 5 chr3 93470543 . AC A 5 chr2 192297664 . T TCCAAAGATA

Dataset 2 (mostly NovaseqX)

22 chr3 93470691 . AT A 12 chr18 110665 . TC T 10 chr5 6039630 . G GTTGT 10 chr5 49599504 . GA G 10 chr2 80565600 . G GAAAC 9 chr9 121816555 . C CGT 9 chr22 47573548 . G GTGTA 9 chr22 37390607 . A AG 8 chr3 93470681 . T TC 8 chr20 15637220 . G GTATTA 7 chr5 79157401 . G GT 7 chr5 24244570 . G GT 7 chr5 117097233 . G GATGT 7 chr4 21851143 . T TGC 7 chr3 93470660 . GC G 7 chr2 235849481 . T TCTC 7 chr18 37633970 . T TGACCTTG 7 chr13 108112528 . A AGATTAC 7 chr12 4848250 . T TTG 7 chr1 218078822 . G GT 6 chr7 155919569 . A AG 6 chr6 92310257 . G GA 6 chr4 143285483 . A ACTC 6 chr21 37727458 . C CGT 6 chr2 129201728 . A ATATG 6 chr12 108979090 . T TATATATATGC 6 chr11 42534858 . G GGT 6 chr10 46545261 . A AT 5 chrX 139912749 . T TTTTTA 5 chr9 116409870 . G GCA 5 chr7 9172174 . A AT 5 chr7 20447188 . C CCT 5 chr7 153920212 . G GAGC 5 chr6 96110820 . TA T 5 chr6 79565540 . T TATC 5 chr3 6183442 . AT A 5 chr3 26930008 . C CTTCCT 5 chr3 198112059 . T TCTGC 5 chr3 113073671 . A AT 5 chr2 20592148 . TG T 5 chr2 182194985 . G GC 5 chr20 31189252 . G GACTCA 5 chr18 72842070 . A AT 5 chr17 71534023 . T TAC 5 chr14 63259340 . A AC 5 chr13 70014560 . T TAAGA 5 chr1 16727339 . A AC 5 chr11 43195703 . C CAT 5 chr11 128162675 . C CGTGTGTGT

You can see one indel artifact region that is in both types of Novaseq -- a chr3 SATELLITE artifact region (chr3:93470362-93470799). I'm assuming you have seen this too. It is not in the standard hg38 NOISE mask.

But you can see for Novaseq X, there is a lager diversity of regions with recurrent indels. We don't see a clear pattern why these regions are doing this.

The issue is that without figuring out the pattern, we don't know how many of the non-recurrent indels are artifacts. Perhaps there are other artifacts hiding among the indels found in only one sample.

Anyway, appreciate hearing what you all figure out about this.

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gevro commented 10 months ago

Thanks!

I agree, probably the reason you are seeing some recurrent SNP artifacts and we are seeing different indel artifacts might be GRCh37 vs hg38. Are these SNP artifacts you sent only seen with NovaseqX? Looking at those regions lifted-over to hg38, I don't see that those regions changed between the assemblies. I wonder why we don't see these artifacts. Maybe if you reanalyze some samples in hg38 you could see if these go away, and if you then see the indel artifacts we see.
We are using v3.2.1. Does this version have the indel bug? If so, we will reprocess with the latest version.
Will there be any change at all in SNP calls if we need to rerun with the latest version, or will only indel calls change? That way we know if we need to redo our SNP analyses.
If we need to upgrade from v3.2.1, do we need to restart from FASTQ, or can we start from the CRAM files previously made with v3.2.1?
If we need to upgrade from v3.2.1, did any parameters change in how we should run the pipeline? Here is what we currently use: dsa: -d 2 -q 30 (+ SNP and NOISE masks) var: -a 50 -b (0 for undiluted nanoseq, 3 for non-nanoseq germline ref) -c 0 -d 2 -f 0.9 -i 1 -m 8 -n 3 -p 0 -q 60 -r 144 -v 0.01 -x 8 -z 15 indel: -a 50 -c 0 -v 0.01 -z 15 --rb 2 --t3 135 --t5 10

gevro commented 10 months ago

Hi, Just checking if you have answers to the above, so that we know if/how we need to rerun with the newer pipeline. Thank you!

fa8sanger commented 10 months ago

Hi, let me go one by one:

I haven’t had time to have a look into this. Not planning right now to try myself on hg38.
Easiest way to verify is to compare the code you are using with: https://github.com/cancerit/NanoSeq/blob/develop/perl/indelCaller_step2.pl Pay attention to lines around 286
The changes do not impact the calling of substitutions, only indels. Changes include a bug fix and additional quality metric outputs for indels

4 and 5. You only need to rerun the indel calling. Those parameter settings look fine to me

On 30 Dec 2023, at 21:17, gevro @.***> wrote:

Thanks!

I agree, probably the reason you are seeing some recurrent SNP artifacts and we are seeing different indel artifacts might be GRCh37 vs hg38. Are these SNP artifacts you sent only seen with NovaseqX? Looking at those regions lifted-over to hg38, I don't see that those regions changed between the assemblies. I wonder why we don't see these artifacts. Maybe if you reanalyze some samples in hg38 you could see if these go away, and if you then see the indel artifacts we see.
We are using v3.2.1. Does this version have the indel bug? If so, we will reprocess with the latest version.
Will there be any change at all in SNP calls if we need to rerun with the latest version, or will only indel calls change? That way we know if we need to redo our SNP analyses.
If we need to upgrade from v3.2.1, do we need to restart from FASTQ, or can we start from the CRAM files previously made with v3.2.1?
If we need to upgrade from v3.2.1, did any parameters change in how we should run the pipeline? Here is what we currently use: dsa: -d 2 -q 30 (+ SNP and NOISE masks) var: -a 50 -b (0 for undiluted nanoseq, 3 for non-nanoseq germline ref) -c 0 -d 2 -f 0.9 -i 1 -m 8 -n 3 -p 0 -q 60 -r 144 -v 0.01 -x 8 -z 15 indel: -a 50 -c 0 -v 0.01 -z 15 --rb 2 --t3 135 --t5 10

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fa8sanger commented 9 months ago

We had a meeting with Illumina yesterday and I passed them this information on potentially artefactual indels. The had a look at some of them in their data and they all look good: either absent or, when present, not showing mapping orientation biases and in perfect linkage when there were nearby SNPs. I wonder if you may have contamination in your samples. It could also be a problem related to the bug in the indel pipeline I guess.

On 29 Dec 2023, at 15:55, gevro @.***> wrote:

Interesting. We didn't see recurrent artifacts in SNPs. How did you identify these, in case we didn't do the search properly?

Regarding indels: upon further analysis, it seems that most of the Novaseq X indel recurrent artifacts we found happen even when the germline reference was also sequenced on Novaseq X. We also found one region with recurrent artifacts for Novaseq samples.

Here are recurrent indels we found in >=5 samples in two different datasets of ~30 independent samples each. One dataset was only Novaseq 6000, and one dataset was mostly Novaseq X.

First column is # of samples in which the indel was detected

Dataset 1 (mostly Novaseq 6000) 28 chr3 93470660 . GC G 19 chr3 93470714 . TTGA T 15 chr3 93470568 . G GA 7 chr3 93470677 . AT A 7 chr3 93470535 . GT G 6 chr3 93470719 . AT A 6 chr3 93470592 . TA T 6 chr3 93470407 . AT A 6 chr1 193234361 . A AAATTAT 5 chr3 93470681 . T TC 5 chr3 93470664 . TG T 5 chr3 93470631 . T TCA 5 chr3 93470543 . AC A 5 chr2 192297664 . T TCCAAAGATA

Dataset 2 (mostly NovaseqX)

22 chr3 93470691 . AT A 12 chr18 110665 . TC T 10 chr5 6039630 . G GTTGT 10 chr5 49599504 . GA G 10 chr2 80565600 . G GAAAC 9 chr9 121816555 . C CGT 9 chr22 47573548 . G GTGTA 9 chr22 37390607 . A AG 8 chr3 93470681 . T TC 8 chr20 15637220 . G GTATTA 7 chr5 79157401 . G GT 7 chr5 24244570 . G GT 7 chr5 117097233 . G GATGT 7 chr4 21851143 . T TGC 7 chr3 93470660 . GC G 7 chr2 235849481 . T TCTC 7 chr18 37633970 . T TGACCTTG 7 chr13 108112528 . A AGATTAC 7 chr12 4848250 . T TTG 7 chr1 218078822 . G GT 6 chr7 155919569 . A AG 6 chr6 92310257 . G GA 6 chr4 143285483 . A ACTC 6 chr21 37727458 . C CGT 6 chr2 129201728 . A ATATG 6 chr12 108979090 . T TATATATATGC 6 chr11 42534858 . G GGT 6 chr10 46545261 . A AT 5 chrX 139912749 . T TTTTTA 5 chr9 116409870 . G GCA 5 chr7 9172174 . A AT 5 chr7 20447188 . C CCT 5 chr7 153920212 . G GAGC 5 chr6 96110820 . TA T 5 chr6 79565540 . T TATC 5 chr3 6183442 . AT A 5 chr3 26930008 . C CTTCCT 5 chr3 198112059 . T TCTGC 5 chr3 113073671 . A AT 5 chr2 20592148 . TG T 5 chr2 182194985 . G GC 5 chr20 31189252 . G GACTCA 5 chr18 72842070 . A AT 5 chr17 71534023 . T TAC 5 chr14 63259340 . A AC 5 chr13 70014560 . T TAAGA 5 chr1 16727339 . A AC 5 chr11 43195703 . C CAT 5 chr11 128162675 . C CGTGTGTGT

You can see one indel artifact region that is in both types of Novaseq -- a chr3 SATELLITE artifact region (chr3:93470362-93470799). I'm assuming you have seen this too. It is not in the standard hg38 NOISE mask.

But you can see for Novaseq X, there is a lager diversity of regions with recurrent indels. We don't see a clear pattern why these regions are doing this.

The issue is that without figuring out the pattern, we don't know how many of the non-recurrent indels are artifacts. Perhaps there are other artifacts hiding among the indels found in only one sample.

Anyway, appreciate hearing what you all figure out about this.

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gevro commented 9 months ago

Thanks for following up with Illumina. I don't think it is contamination because FREEMIX values were fine and these artifacts were recurrent across several samples that were prepared in batches on different days. But we are rerunning with the v3.5.4 pipeline and hopefully that will mitigate or resolve the issue.

gevro commented 8 months ago

Hi, Just to update: now that we reran with v3.5.5 of your pipeline, we still see this indel artifact issue.

For now we are mitigating this by filtering out indels found in > X samples among a cohort of samples, where we set X to a number that makes sense depending on the size of the project cohort.

gevro commented 8 months ago

And just to add, we're also seeing such artifactual single-base substitutions specifically in NovaseqX samples that we sequenced more deeply where the matched germline was sequenced on Novaseq 6000.

fa8sanger commented 8 months ago

Thanks for letting me know. Regarding the indels, that’s strange, we haven’t seen that problem. Perhaps worth digging into those indels more. If you send me their final VCF I could have a look. The latest version provides some additional quality metrics (NM, ASXS, etc) which may be helpful

On 14 Feb 2024, at 04:42, gevro @.***> wrote:

And just to add, we're also seeing such artifactual single-base substitutions specifically in NovaseqX samples that we sequenced more deeply where the matched germline was sequenced on Novaseq 6000.

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gevro commented 8 months ago

Sure, here are the VCF lines of the most recurrent SNP artifact and the most recurrent indel artifact. This is from mouse genome (mm10). I also attached IGV plots for each showing from top to bottom two germline samples (Novaseq 6000), two somatic samples (Novaseq X) and one somatic sample (Novaseq 6000).

It looks like clearly differential sequencing artifacts between the instruments. But you can see something interesting. For the SNP artifact, it is because Novaseq X has sequencing artifacts that are not in the Novaseq 6000 germline reference, whereas for the indel artifact it is the opposite situation.

Would be happy to hear any suggestions for how to more systematically remove these, without filtering out a lot of the genome. For now we are just going to identify them as 'recurrent' artifacts across more than a certain fraction of samples.

Recurrent somatic SNP

sample 11
chr5    66250784    .   C   T   .   PASS    BTAG=GCG|GTT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=6;DEPTH_REV=3;DEPTH_NORM_FWD=55;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=121;BULK_NM=0

sample 14
chr5    66250784    .   C   T   .   PASS    BTAG=AGA|CGC;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=51;DEPTH_NORM_REV=0;DPLX_ASXS=77;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=GAA|TTT;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=8;DEPTH_NORM_FWD=51;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 28
chr5    66250784    .   C   T   .   PASS    BTAG=TCA|CAG;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=2;DEPTH_NORM_FWD=35;DEPTH_NORM_REV=0;DPLX_ASXS=83;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=120;BULK_NM=0
chr5    66250784    .   C   T   .   PASS    BTAG=GAG|GGG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=35;DEPTH_NORM_REV=0;DPLX_ASXS=75;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=120;BULK_NM=0
chr5    66250784    .   C   T   .   PASS    BTAG=CCC|GAT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=35;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=120;BULK_NM=0

sample 8
chr5    66250784    .   C   T   .   PASS    BTAG=CTA|ACA;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=51;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=120;BULK_NM=1

sample 13
chr5    66250784    .   C   T   .   PASS    BTAG=GAT|ACC;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=3;DEPTH_REV=7;DEPTH_NORM_FWD=48;DEPTH_NORM_REV=0;DPLX_ASXS=81;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=120;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=ATG|GAC;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=48;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=120;BULK_NM=1

sample 33
chr5    66250784    .   C   T   .   PASS    BTAG=CCA|CCT;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=28;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=120;BULK_NM=1

sample 27
chr5    66250784    .   C   T   .   PASS    BTAG=AAG|AAG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=43;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=118;BULK_NM=1

sample 36
chr5    66250784    .   C   T   .   PASS    BTAG=TTT|TTC;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=41;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=117;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=TAG|AGT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=6;DEPTH_REV=4;DEPTH_NORM_FWD=41;DEPTH_NORM_REV=0;DPLX_ASXS=83;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=117;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=TCC|GCT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=41;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=117;BULK_NM=1

sample 6
chr5    66250784    .   C   T   .   PASS    BTAG=CCA|ATT;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=3;DEPTH_REV=3;DEPTH_NORM_FWD=62;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=119;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=CCG|CTA;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=62;DEPTH_NORM_REV=0;DPLX_ASXS=77;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=119;BULK_NM=1

sample 26
chr5    66250784    .   C   T   .   PASS    BTAG=TCT|TAA;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=3;DEPTH_REV=3;DEPTH_NORM_FWD=29;DEPTH_NORM_REV=0;DPLX_ASXS=81;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=119;BULK_NM=1

sample 31
chr5    66250784    .   C   T   .   PASS    BTAG=CAG|TGC;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=24;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 23
chr5    66250784    .   C   T   .   PASS    BTAG=CGC|GCT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=5;DEPTH_REV=4;DEPTH_NORM_FWD=73;DEPTH_NORM_REV=0;DPLX_ASXS=77;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=121;BULK_NM=1

sample 35
chr5    66250784    .   C   T   .   PASS    BTAG=TTA|CTG;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=45;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=118;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=GGC|TAC;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=5;DEPTH_REV=10;DEPTH_NORM_FWD=45;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=118;BULK_NM=1

sample 3
chr5    66250784    .   C   T   .   PASS    BTAG=AGG|GGT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=6;DEPTH_REV=2;DEPTH_NORM_FWD=45;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=116;BULK_NM=1

sample 34
chr5    66250784    .   C   T   .   PASS    BTAG=CAA|GCG;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=6;DEPTH_REV=4;DEPTH_NORM_FWD=46;DEPTH_NORM_REV=0;DPLX_ASXS=85;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=CAC|GGG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=11;DEPTH_REV=6;DEPTH_NORM_FWD=46;DEPTH_NORM_REV=0;DPLX_ASXS=82;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=GCC|AAG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=6;DEPTH_REV=5;DEPTH_NORM_FWD=46;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=119;BULK_NM=1

sample 29
chr5    66250784    .   C   T   .   PASS    BTAG=AGC|TAA;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=34;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=CTG|GAT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=34;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=TTG|GTT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=34;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 7
chr5    66250784    .   C   T   .   PASS    BTAG=CTC|TGG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=56;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=121;BULK_NM=0

sample 4
chr5    66250784    .   C   T   .   PASS    BTAG=ACA|GGC;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=5;DEPTH_REV=4;DEPTH_NORM_FWD=41;DEPTH_NORM_REV=0;DPLX_ASXS=83;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=114;BULK_NM=1

sample 16
chr5    66250784    .   C   T   .   PASS    BTAG=GAG|AGG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=79;DEPTH_NORM_REV=0;DPLX_ASXS=81;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=120;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=ATC|TGT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=79;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=120;BULK_NM=1

sample 15
chr5    66250784    .   C   T   .   PASS    BTAG=ACC|GAA;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=32;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=121;BULK_NM=0

sample 10
chr5    66250784    .   C   T   .   PASS    BTAG=TTT|CTA;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=62;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 32
chr5    66250784    .   C   T   .   PASS    BTAG=ATC|AGT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=25;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1
chr5    66250784    .   C   T   .   PASS    BTAG=TTC|TGA;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=6;DEPTH_NORM_FWD=25;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 19
chr5    66250784    .   C   T   .   PASS    BTAG=CGC|CCT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=5;DEPTH_REV=5;DEPTH_NORM_FWD=44;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=118;BULK_NM=1

Recurrent somatic indel

sample 9
chr16   9152718 .   GC  G   48.4186 PASS    INDEL;IDV=7;IMF=1;DP=7;VDB=0.000137386;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,TCC,TTA;BBEG=9152627;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=86;DPLX_CLIP=0;DPLX_NM=4.5;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4ir=1:105,27:7:7:0:0,0,7,0
chr16   9152718 .   GC  G   59.4184 PASS    INDEL;IDV=7;IMF=1;DP=7;VDB=6.71664e-05;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GTA,CTA;BBEG=9152626;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=88;DPLX_CLIP=0;DPLX_NM=4.7;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4i-m1:110,21:7:7:0:0,0,7,0
chr16   9152718 .   GC  G   86.3149 PASS    INDEL;IDV=10;IMF=0.909091;DP=11;VDB=6.99472e-07;SGB=-0.670168;RPBZ=-3.16228;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=-3.16228;MQ0F=0;AC=1;AN=1;DP4=1,0,10,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,ATA,TTG;BBEG=9152626;BEND=9152991;DEPTH_FWD=7;DEPTH_REV=4;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=96;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCCnoseq_GT:PL:DP:DV:SP:DP4utput=1:113,0:11:10:0:1,0,10,0r.bed
chr16   9152718 .   GC  G   58.171  PASS    INDEL;IDV=12;IMF=0.923077;DP=13;VDB=1.40522e-07;SGB=-0.680642;RPBZ=0.543557;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0.543557;MQ0F=0;AC=1;AN=1;DP4=1,0,12,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,TGA,TAT;BBEG=9152626;BEND=9152992;DEPTH_FWD=8;DEPTH_REV=5;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=93;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:93,8:13:12:0:1,0,12,0
chr16   9152718 .   GC  G   43.4191 PASS    INDEL;IDV=7;IMF=1;DP=7;VDB=0.000207967;SGB=-0.636426;BQBZ=-1.1547;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GGA,CAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=83;DPLX_CLIP=0;DPLX_NM=5.3;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:73,0:7:7:0:0,0,7,0
chr16   9152718 .   GC  G   42.4147 PASS    INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,GTG,GGT;BBEG=9152627;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=91;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4  1:72,0:4:4:0:0,0,4,0
chr16   9152718 .   GC  G   64.4147 PASS    INDEL;IDV=7;IMF=1;DP=7;VDB=0.000157941;SGB=-0.636426;BQBZ=-1.87083;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,AGA,CAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=5;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=87;DPLX_CLIP=0;DPLX_NM=5.5;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:94,0:7:7:0:0,0,7,0
chr16   9152718 .   GC  G   73.4149 PASS    INDEL;IDV=8;IMF=1;DP=8;VDB=2.75158e-05;SGB=-0.651104;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,8,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,GTC,AAA;BBEG=9152627;BEND=9152992;DEPTH_FWD=5;DEPTH_REV=3;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=94;DPLX_CLIP=0;DPLX_NM=4.2;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4  1:103,0:8:8:0:0,0,8,0

sample 36
chr16   9152718 .   GC  G   52.4146 PASS    INDEL;IDV=6;IMF=1;DP=6;VDB=0.000411984;SGB=-0.616816;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,6,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GCT,ATC;BBEG=9152626;BEND=9152991;DEPTH_FWD=4;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=82;DPLX_CLIP=0;DPLX_NM=6.5;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4  1:82,0:6:6:0:0,0,6,0
chr16   9152718 .   GC  G   86.415  PASS    INDEL;IDV=8;IMF=1;DP=8;VDB=2.75158e-05;SGB=-0.651104;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,8,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,TAA,AAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=100;DPLX_CLIP=0;DPLX_NM=1.7;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:116,0:8:8:0:0,0,8,0
chr16   9152718 .   GC  G   73.4149 PASS    INDEL;IDV=8;IMF=1;DP=8;VDB=1.33331e-05;SGB=-0.651104;BQBZ=-1.76383;MQ0F=0;AC=1;AN=1;DP4=0,0,8,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GCA,CCA;BBEG=9152626;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=93;DPLX_CLIP=0;DPLX_NM=3.8;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:103,0:8:8:0:0,0,8,0
chr16   9152718 .   GC  G   34.4235 PASS    INDEL;IDV=6;IMF=1;DP=6;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.73205;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GTA,ACG;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=63;DPLX_CLIP=0;DPLX_NM=11.5;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:90,26:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   91.4151 PASS    INDEL;IDV=11;IMF=1;DP=11;VDB=5.51542e-08;SGB=-0.676189;BQBZ=-1.87617;MQ0F=0;AC=1;AN=1;DP4=0,0,11,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,CTG,TCC;BBEG=9152627;BEND=9152991;DEPTH_FWD=7;DEPTH_REV=4;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=96;DPLX_CLIP=0;DPLX_NM=2.6;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:121,0:11:11:0:0,0,11,0
chr16   9152718 .   GC  G   35.4156 PASS    INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;BQBZ=-0.471405;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,AGA,TAA;BBEG=9152626;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=75;DPLX_CLIP=0;DPLX_NM=7.5;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:65,0:4:4:0:0,0,4,0
chr16   9152718 .   GC  G   18.5854 PASS    INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=92;RB=chr16,9152626,9153567,TAT,TTA;BBEG=9152626;BEND=9153567;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=92;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4  1:74,26:4:4:0:0,0,4,0
chr16   9152718 .   GC  G   58.4147 PASS    INDEL;IDV=4;IMF=0.8;DP=5;VDB=0.00187095;SGB=-0.590765;RPBZ=0;MQBZ=0;BQBZ=-1.58114;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,TAA,CTT;BBEG=9152626;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=93;DPLX_CLIP=0;DPLX_NM=3.5;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:88,0:5:5:0:0,0,5,0

sample 6
chr16   9152718 .   GC  G   99.3784 PASS    INDEL;IDV=16;IMF=0.941176;DP=17;VDB=1.29641e-11;SGB=-0.689466;RPBZ=-4;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=-4;MQ0F=0;AC=1;AN=1;DP4=1,0,16,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GCA,ACC;BBEG=9152626;BEND=9152991;DEPTH_FWD=11;DEPTH_REV=6;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=98;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=60;BULK_NM=1;NN=[0:154:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:126,0:17:16:0:1,0,16,0
chr16   9152718 .   GC  G   46.4184 PASS    INDEL;IDV=7;IMF=1;DP=7;VDB=0.000311348;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CTA,GAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=5;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=84;DPLX_CLIP=0;DPLX_NM=5;BULK_ASXS=60;BULK_NM=1;NN=[0:154:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:93,17:7:7:0:0,0,7,0
chr16   9152718 .   GC  G   99.3986 PASS    INDEL;IDV=14;IMF=0.933333;DP=15;VDB=2.26006e-08;SGB=-0.686358;RPBZ=-3.74166;MQBZ=0;BQBZ=-1.45444;SCBZ=-3.74166;MQ0F=0;AC=1;AN=1;DP4=1,0,14,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,AAC,ATT;BBEG=9152627;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=11;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=87;DPLX_CLIP=0;DPLX_NM=3.4;BULK_ASXS=60;BULK_NM=1;NN=[0:154:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:126,0:15:14:0:1,0,14,0

sample 7
chr16   9152718 .   GC  G   55.4146 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,AAC,AAG;BBEG=9152627;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=88;DPLX_CLIP=0;DPLX_NM=4.5;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:85,0:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   47.4146 PASS    INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,TTA,TAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=90;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4  1:77,0:4:4:0:0,0,4,0
chr16   9152718 .   GC  G   31.4276 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.49071;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CGA,GTC;BBEG=9152626;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=86;DPLX_CLIP=0;DPLX_NM=5.3;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:86,25:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   18.4763 PASS    INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;BQBZ=-1;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,TAG,GTT;BBEG=9152627;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=72;DPLX_CLIP=0;DPLX_NM=8.5;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:48,0:4:4:0:0,0,4,0
chr16   9152718 .   GC  G   57.4183 PASS    INDEL;IDV=7;IMF=1;DP=7;VDB=6.71664e-05;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GTT,TAG;BBEG=9152626;BEND=9152991;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=96;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:110,23:7:7:0:0,0,7,0

sample 10
chr16   9152718 .   GC  G   31.4548 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,TGG,ATA;BBEG=9152627;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=84;DPLX_CLIP=0;DPLX_NM=6;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:93,32:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   61.1452 PASS    INDEL;IDV=11;IMF=0.916667;DP=12;VDB=2.26006e-08;SGB=-0.676189;RPBZ=0;MQBZ=0;BQBZ=-3.31662;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=1,0,11,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CTT,AAG;BBEG=9152626;BEND=9152991;DEPTH_FWD=5;DEPTH_REV=7;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=101;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:131,43:12:11:0:1,0,11,0
chr16   9152718 .   GC  G   51.2953 PASS    INDEL;IDV=9;IMF=0.9;DP=10;VDB=1.02904e-05;SGB=-0.662043;RPBZ=-1.93649;MQBZ=0;BQBZ=-2.23607;SCBZ=-1.93649;MQ0F=0;AC=1;AN=1;DP4=1,0,9,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,TAA,GAG;BBEG=9152626;BEND=9152991;DEPTH_FWD=5;DEPTH_REV=5;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=5.2;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:78,0:10:9:0:1,0,9,0
chr16   9152718 .   GC  G   78.3164 PASS    INDEL;IDV=10;IMF=0.909091;DP=11;VDB=6.99472e-07;SGB=-0.670168;RPBZ=0;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=1,0,10,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,TAG,AAA;BBEG=9152627;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=7;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=94;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:121,16:11:10:0:1,0,10,0
chr16   9152718 .   GC  G   27.5107 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00558876;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,GGG,TCA;BBEG=9152627;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=75;DPLX_CLIP=0;DPLX_NM=5.5;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:89,32:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   97.4152 PASS    INDEL;IDV=15;IMF=1;DP=15;VDB=8.40038e-10;SGB=-0.688148;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,15,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,CAG,CGA;BBEG=9152627;BEND=9152992;DEPTH_FWD=8;DEPTH_REV=7;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:127,0:15:15:0:0,0,15,0
chr16   9152718 .   GC  G   75.2638 PASS    INDEL;IDV=9;IMF=0.9;DP=10;VDB=5.52906e-06;SGB=-0.662043;RPBZ=0;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=1,0,9,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GAT,TAA;BBEG=9152626;BEND=9152992;DEPTH_FWD=6;DEPTH_REV=4;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4  1:102,0:10:9:0:1,0,9,0

sample 32
chr16   9152718 .   GC  G   51.4182 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,AAT,AGT;BBEG=9152626;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=100;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4  1:110,29:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   60.4147 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00248765;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CAA,GGG;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=92;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:90,0:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   40.4249 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.58114;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,AGA,TCT;BBEG=9152626;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=94;DPLX_CLIP=0;DPLX_NM=3.7;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:104,34:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   77.4149 PASS    INDEL;IDV=8;IMF=1;DP=8;VDB=1.33331e-05;SGB=-0.651104;BQBZ=-2;MQ0F=0;AC=1;AN=1;DP4=0,0,8,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GGT,TTA;BBEG=9152626;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:107,0:8:8:0:0,0,8,0
chr16   9152718 .   GC  G   68.4148 PASS    INDEL;IDV=6;IMF=1;DP=6;VDB=0.000560625;SGB=-0.616816;BQBZ=-2.23607;MQ0F=0;AC=1;AN=1;DP4=0,0,6,0;MQ=60;QPOS=92;RB=chr16,9152626,9153566,TGT,TAG;BBEG=9152626;BEND=9153566;DEPTH_FWD=4;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:98,0:6:6:0:0,0,6,0
chr16   9152718 .   GC  G   72.4187 PASS    INDEL;IDV=10;IMF=1;DP=10;VDB=6.99472e-07;SGB=-0.670168;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,10,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GCA,TAA;BBEG=9152626;BEND=9152992;DEPTH_FWD=7;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=91;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC   GT:PL:DP:DV:SP:DP4  1:121,19:10:10:0:0,0,10,0
chr16   9152718 .   GC  G   47.4146 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00248765;SGB=-0.590765;BQBZ=-2;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GGT,AAT;BBEG=9152626;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=93;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:77,0:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   41.4185 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.73205;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CTT,CTG;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=92;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC    GT:PL:DP:DV:SP:DP4  1:84,13:5:5:0:0,0,5,0
chr16   9152718 .   GC  G   69.4148 PASS    INDEL;IDV=7;IMF=1;DP=7;VDB=0.00011279;SGB=-0.636426;BQBZ=-1.87083;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,GAC,TGA;BBEG=9152627;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=81;DPLX_CLIP=0;DPLX_NM=5.8;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:99,0:7:7:0:0,0,7,0
chr16   9152718 .   GC  G   60.4147 PASS    INDEL;IDV=6;IMF=1;DP=6;VDB=0.000411984;SGB=-0.616816;BQBZ=-1.58114;MQ0F=0;AC=1;AN=1;DP4=0,0,6,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,AGG,CCC;BBEG=9152627;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=91;DPLX_CLIP=0;DPLX_NM=4.3;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:90,0:6:6:0:0,0,6,0
chr16   9152718 .   GC  G   60.218  PASS    INDEL;IDV=10;IMF=0.909091;DP=11;VDB=6.99472e-07;SGB=-0.670168;RPBZ=0;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=1,0,10,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,GAG,AGG;BBEG=9152627;BEND=9152991;DEPTH_FWD=7;DEPTH_REV=4;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=94;DPLX_CLIP=0;DPLX_NM=3.5;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:145,58:11:10:0:1,0,10,0
chr16   9152718 .   GC  G   62.4147 PASS    INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.49071;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,TCG,GTC;BBEG=9152627;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=103;DPLX_CLIP=0;DPLX_NM=1.7;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:92,0:5:5:0:0,0,5,0
chr16   9152718 .   GCC G   70.4187 PASS    INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;BQBZ=-1.73205;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,CCA,GTA;BBEG=9152626;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=98;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:115,15:4:4:0:0,0,4,0

sample 22
chr16   9152718 .   GC  G   51.4183 PASS    INDEL;IDV=7;IMF=1;DP=7;VDB=9.50611e-05;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GTT,ACG;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=5;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=91;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=61;BULK_NM=1;NN=[0:157:0];SEQ=ATAAAGATGCCCCCCCCC  GT:PL:DP:DV:SP:DP4  1:104,23:7:7:0:0,0,7,0
chr16   9152718 .   GCC G   95.3043 PASS    INDEL;IDV=13;IMF=0.928571;DP=14;VDB=3.3258e-08;SGB=-0.683931;RPBZ=0.27735;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0.27735;MQ0F=0;AC=1;AN=1;DP4=1,0,13,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,ATA,ATC;BBEG=9152626;BEND=9152991;DEPTH_FWD=4;DEPTH_REV=10;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=98;DPLX_CLIP=0;DPLX_NM=2.7;BULK_ASXS=61;BULK_NM=1;NN=[0:157:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4  1:208,86:14:13:0:1,0,13,0

fa8sanger commented 8 months ago

Regarding the substitution, there is a very strong read position bias (QPOS=107/108). That’s expected if you are fragmenting the genome with the restriction enzyme (standard NanoSeq). If you are using some random fragmentation, a strong position bias is a sign of artifacts, usually happens because there’s a nearby indel and the aligner prefers to introduce a mismatch. These are solved with indel realignment. We don’t do that because it is very costly computationally.

Regarding the indels, there is a very strong mapping orientation bias (DEPTH_NORM_REV=0). Again, this is expected if the matched normals are prepared with the restriction enzyme. Otherwise it’s a bad sign. I don’t see any other problem.

Mouse being a non-human species would work worse without SNP/NOISE masks. You could try to create one if you haven’t already.

Regarding the IGV screenshot, it seems NS6000 shows a lot of soft-clipping while NSX shows indels there. I don’t know why. Again you could protect yourself by using SNP/NOISE masks.

On 15 Feb 2024, at 01:32, gevro @.***> wrote:

Sure, here are the VCF lines of the most recurrent SNP artifact and the most recurrent indel artifact. This is from mouse genome (mm10). I also attached IGV plots for each showing from top to bottom two germline samples (Novaseq 6000), two somatic samples (Novaseq X) and one somatic sample (Novaseq 6000).

It looks like clearly differential sequencing artifacts between the instruments. But you can see something interesting. For the SNP artifact, it is because Novaseq X has sequencing artifacts that are not in the Novaseq 6000 germline reference, whereas for the indel artifact it is the opposite situation.

Would be happy to hear any suggestions for how to more systematically remove these, without filtering out a lot of the genome. For now we are just going to identify them as 'recurrent' artifacts across more than a certain fraction of samples.

Recurrent somatic SNP

sample 11 chr5 66250784 . C T . PASS BTAG=GCG|GTT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=6;DEPTH_REV=3;DEPTH_NORM_FWD=55;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=121;BULK_NM=0

sample 14 chr5 66250784 . C T . PASS BTAG=AGA|CGC;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=51;DEPTH_NORM_REV=0;DPLX_ASXS=77;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=GAA|TTT;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=8;DEPTH_NORM_FWD=51;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 28 chr5 66250784 . C T . PASS BTAG=TCA|CAG;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=2;DEPTH_NORM_FWD=35;DEPTH_NORM_REV=0;DPLX_ASXS=83;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=120;BULK_NM=0 chr5 66250784 . C T . PASS BTAG=GAG|GGG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=35;DEPTH_NORM_REV=0;DPLX_ASXS=75;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=120;BULK_NM=0 chr5 66250784 . C T . PASS BTAG=CCC|GAT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=35;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=120;BULK_NM=0

sample 8 chr5 66250784 . C T . PASS BTAG=CTA|ACA;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=51;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=120;BULK_NM=1

sample 13 chr5 66250784 . C T . PASS BTAG=GAT|ACC;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=3;DEPTH_REV=7;DEPTH_NORM_FWD=48;DEPTH_NORM_REV=0;DPLX_ASXS=81;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=120;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=ATG|GAC;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=48;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=120;BULK_NM=1

sample 33 chr5 66250784 . C T . PASS BTAG=CCA|CCT;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=28;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=120;BULK_NM=1

sample 27 chr5 66250784 . C T . PASS BTAG=AAG|AAG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=43;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=118;BULK_NM=1

sample 36 chr5 66250784 . C T . PASS BTAG=TTT|TTC;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=41;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=117;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=TAG|AGT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=6;DEPTH_REV=4;DEPTH_NORM_FWD=41;DEPTH_NORM_REV=0;DPLX_ASXS=83;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=117;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=TCC|GCT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=41;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=117;BULK_NM=1

sample 6 chr5 66250784 . C T . PASS BTAG=CCA|ATT;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=3;DEPTH_REV=3;DEPTH_NORM_FWD=62;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=119;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=CCG|CTA;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=62;DEPTH_NORM_REV=0;DPLX_ASXS=77;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=119;BULK_NM=1

sample 26 chr5 66250784 . C T . PASS BTAG=TCT|TAA;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=3;DEPTH_REV=3;DEPTH_NORM_FWD=29;DEPTH_NORM_REV=0;DPLX_ASXS=81;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=119;BULK_NM=1

sample 31 chr5 66250784 . C T . PASS BTAG=CAG|TGC;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=24;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 23 chr5 66250784 . C T . PASS BTAG=CGC|GCT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=5;DEPTH_REV=4;DEPTH_NORM_FWD=73;DEPTH_NORM_REV=0;DPLX_ASXS=77;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=121;BULK_NM=1

sample 35 chr5 66250784 . C T . PASS BTAG=TTA|CTG;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=45;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=118;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=GGC|TAC;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=5;DEPTH_REV=10;DEPTH_NORM_FWD=45;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=118;BULK_NM=1

sample 3 chr5 66250784 . C T . PASS BTAG=AGG|GGT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=6;DEPTH_REV=2;DEPTH_NORM_FWD=45;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=116;BULK_NM=1

sample 34 chr5 66250784 . C T . PASS BTAG=CAA|GCG;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=6;DEPTH_REV=4;DEPTH_NORM_FWD=46;DEPTH_NORM_REV=0;DPLX_ASXS=85;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=CAC|GGG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=11;DEPTH_REV=6;DEPTH_NORM_FWD=46;DEPTH_NORM_REV=0;DPLX_ASXS=82;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=GCC|AAG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=6;DEPTH_REV=5;DEPTH_NORM_FWD=46;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=119;BULK_NM=1

sample 29 chr5 66250784 . C T . PASS BTAG=AGC|TAA;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=34;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=CTG|GAT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=34;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=TTG|GTT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=34;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 7 chr5 66250784 . C T . PASS BTAG=CTC|TGG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=56;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=121;BULK_NM=0

sample 4 chr5 66250784 . C T . PASS BTAG=ACA|GGC;BBEG=66250676;BEND=66251076;TRI=TCT>T;QPOS=108;DEPTH_FWD=5;DEPTH_REV=4;DEPTH_NORM_FWD=41;DEPTH_NORM_REV=0;DPLX_ASXS=83;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=114;BULK_NM=1

sample 16 chr5 66250784 . C T . PASS BTAG=GAG|AGG;BBEG=66250677;BEND=66251076;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=5;DEPTH_NORM_FWD=79;DEPTH_NORM_REV=0;DPLX_ASXS=81;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=120;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=ATC|TGT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=79;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=120;BULK_NM=1

sample 15 chr5 66250784 . C T . PASS BTAG=ACC|GAA;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=32;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=121;BULK_NM=0

sample 10 chr5 66250784 . C T . PASS BTAG=TTT|CTA;BBEG=66250676;BEND=66251077;TRI=TCT>T;QPOS=108;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=62;DEPTH_NORM_REV=0;DPLX_ASXS=80;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 32 chr5 66250784 . C T . PASS BTAG=ATC|AGT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=25;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1 chr5 66250784 . C T . PASS BTAG=TTC|TGA;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=4;DEPTH_REV=6;DEPTH_NORM_FWD=25;DEPTH_NORM_REV=0;DPLX_ASXS=79;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=119;BULK_NM=1

sample 19 chr5 66250784 . C T . PASS BTAG=CGC|CCT;BBEG=66250677;BEND=66251077;TRI=TCT>T;QPOS=107;DEPTH_FWD=5;DEPTH_REV=5;DEPTH_NORM_FWD=44;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=118;BULK_NM=1

Screenshot.2024-02-14.at.8.23.13.PM.png (view on web) [github.com]https://urldefense.proofpoint.com/v2/url?u=https-3A__github.com_cancerit_NanoSeq_assets_47928628_ca2a9b3d-2D85eb-2D4b16-2D89b7-2Dffd5fc3e6ea7&d=DwMCaQ&c=D7ByGjS34AllFgecYw0iC6Zq7qlm8uclZFI0SqQnqBo&r=v9-R7fUmjpv-9Zaqyk1nlnlOC3qPkTEJz5tyYxg2uec&m=N33UpN2-HaUrdhoPZvhfmU6DpCXSKrc9ecSZQn4B4ZPLEMbqXE6aVFbnf1TYmVnB&s=E6V49v9qR04FQUtzPZ7OYhgASSDn_Gt2moTI5t-xNSo&e=

Recurrent somatic indel

sample 9 chr16 9152718 . GC G 48.4186 PASS INDEL;IDV=7;IMF=1;DP=7;VDB=0.000137386;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,TCC,TTA;BBEG=9152627;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=86;DPLX_CLIP=0;DPLX_NM=4.5;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4ir=1:105,27:7:7:0:0,0,7,0 chr16 9152718 . GC G 59.4184 PASS INDEL;IDV=7;IMF=1;DP=7;VDB=6.71664e-05;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GTA,CTA;BBEG=9152626;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=88;DPLX_CLIP=0;DPLX_NM=4.7;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4i-m1:110,21:7:7:0:0,0,7,0 chr16 9152718 . GC G 86.3149 PASS INDEL;IDV=10;IMF=0.909091;DP=11;VDB=6.99472e-07;SGB=-0.670168;RPBZ=-3.16228;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=-3.16228;MQ0F=0;AC=1;AN=1;DP4=1,0,10,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,ATA,TTG;BBEG=9152626;BEND=9152991;DEPTH_FWD=7;DEPTH_REV=4;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=96;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCCnoseq_GT:PL:DP:DV:SP:DP4utput=1:113,0:11:10:0:1,0,10,0r.bed chr16 9152718 . GC G 58.171 PASS INDEL;IDV=12;IMF=0.923077;DP=13;VDB=1.40522e-07;SGB=-0.680642;RPBZ=0.543557;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0.543557;MQ0F=0;AC=1;AN=1;DP4=1,0,12,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,TGA,TAT;BBEG=9152626;BEND=9152992;DEPTH_FWD=8;DEPTH_REV=5;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=93;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:93,8:13:12:0:1,0,12,0 chr16 9152718 . GC G 43.4191 PASS INDEL;IDV=7;IMF=1;DP=7;VDB=0.000207967;SGB=-0.636426;BQBZ=-1.1547;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GGA,CAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=4;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=83;DPLX_CLIP=0;DPLX_NM=5.3;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:73,0:7:7:0:0,0,7,0 chr16 9152718 . GC G 42.4147 PASS INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,GTG,GGT;BBEG=9152627;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=91;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:72,0:4:4:0:0,0,4,0 chr16 9152718 . GC G 64.4147 PASS INDEL;IDV=7;IMF=1;DP=7;VDB=0.000157941;SGB=-0.636426;BQBZ=-1.87083;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,AGA,CAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=5;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=87;DPLX_CLIP=0;DPLX_NM=5.5;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:94,0:7:7:0:0,0,7,0 chr16 9152718 . GC G 73.4149 PASS INDEL;IDV=8;IMF=1;DP=8;VDB=2.75158e-05;SGB=-0.651104;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,8,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,GTC,AAA;BBEG=9152627;BEND=9152992;DEPTH_FWD=5;DEPTH_REV=3;DEPTH_NORM_FWD=15;DEPTH_NORM_REV=0;DPLX_ASXS=94;DPLX_CLIP=0;DPLX_NM=4.2;BULK_ASXS=61;BULK_NM=1;NN=[0:147:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:103,0:8:8:0:0,0,8,0

sample 36 chr16 9152718 . GC G 52.4146 PASS INDEL;IDV=6;IMF=1;DP=6;VDB=0.000411984;SGB=-0.616816;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,6,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GCT,ATC;BBEG=9152626;BEND=9152991;DEPTH_FWD=4;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=82;DPLX_CLIP=0;DPLX_NM=6.5;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:82,0:6:6:0:0,0,6,0 chr16 9152718 . GC G 86.415 PASS INDEL;IDV=8;IMF=1;DP=8;VDB=2.75158e-05;SGB=-0.651104;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,8,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,TAA,AAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=100;DPLX_CLIP=0;DPLX_NM=1.7;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:116,0:8:8:0:0,0,8,0 chr16 9152718 . GC G 73.4149 PASS INDEL;IDV=8;IMF=1;DP=8;VDB=1.33331e-05;SGB=-0.651104;BQBZ=-1.76383;MQ0F=0;AC=1;AN=1;DP4=0,0,8,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GCA,CCA;BBEG=9152626;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=5;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=93;DPLX_CLIP=0;DPLX_NM=3.8;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:103,0:8:8:0:0,0,8,0 chr16 9152718 . GC G 34.4235 PASS INDEL;IDV=6;IMF=1;DP=6;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.73205;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GTA,ACG;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=63;DPLX_CLIP=0;DPLX_NM=11.5;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:90,26:5:5:0:0,0,5,0 chr16 9152718 . GC G 91.4151 PASS INDEL;IDV=11;IMF=1;DP=11;VDB=5.51542e-08;SGB=-0.676189;BQBZ=-1.87617;MQ0F=0;AC=1;AN=1;DP4=0,0,11,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,CTG,TCC;BBEG=9152627;BEND=9152991;DEPTH_FWD=7;DEPTH_REV=4;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=96;DPLX_CLIP=0;DPLX_NM=2.6;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:121,0:11:11:0:0,0,11,0 chr16 9152718 . GC G 35.4156 PASS INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;BQBZ=-0.471405;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,AGA,TAA;BBEG=9152626;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=75;DPLX_CLIP=0;DPLX_NM=7.5;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:65,0:4:4:0:0,0,4,0 chr16 9152718 . GC G 18.5854 PASS INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=92;RB=chr16,9152626,9153567,TAT,TTA;BBEG=9152626;BEND=9153567;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=92;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:74,26:4:4:0:0,0,4,0 chr16 9152718 . GC G 58.4147 PASS INDEL;IDV=4;IMF=0.8;DP=5;VDB=0.00187095;SGB=-0.590765;RPBZ=0;MQBZ=0;BQBZ=-1.58114;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,TAA,CTT;BBEG=9152626;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=93;DPLX_CLIP=0;DPLX_NM=3.5;BULK_ASXS=61;BULK_NM=1;NN=[0:187:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:88,0:5:5:0:0,0,5,0

sample 6 chr16 9152718 . GC G 99.3784 PASS INDEL;IDV=16;IMF=0.941176;DP=17;VDB=1.29641e-11;SGB=-0.689466;RPBZ=-4;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=-4;MQ0F=0;AC=1;AN=1;DP4=1,0,16,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GCA,ACC;BBEG=9152626;BEND=9152991;DEPTH_FWD=11;DEPTH_REV=6;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=98;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=60;BULK_NM=1;NN=[0:154:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:126,0:17:16:0:1,0,16,0 chr16 9152718 . GC G 46.4184 PASS INDEL;IDV=7;IMF=1;DP=7;VDB=0.000311348;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CTA,GAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=5;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=84;DPLX_CLIP=0;DPLX_NM=5;BULK_ASXS=60;BULK_NM=1;NN=[0:154:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:93,17:7:7:0:0,0,7,0 chr16 9152718 . GC G 99.3986 PASS INDEL;IDV=14;IMF=0.933333;DP=15;VDB=2.26006e-08;SGB=-0.686358;RPBZ=-3.74166;MQBZ=0;BQBZ=-1.45444;SCBZ=-3.74166;MQ0F=0;AC=1;AN=1;DP4=1,0,14,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,AAC,ATT;BBEG=9152627;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=11;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=87;DPLX_CLIP=0;DPLX_NM=3.4;BULK_ASXS=60;BULK_NM=1;NN=[0:154:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:126,0:15:14:0:1,0,14,0

sample 7 chr16 9152718 . GC G 55.4146 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,AAC,AAG;BBEG=9152627;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=88;DPLX_CLIP=0;DPLX_NM=4.5;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:85,0:5:5:0:0,0,5,0 chr16 9152718 . GC G 47.4146 PASS INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,TTA,TAC;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=90;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:77,0:4:4:0:0,0,4,0 chr16 9152718 . GC G 31.4276 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.49071;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CGA,GTC;BBEG=9152626;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=86;DPLX_CLIP=0;DPLX_NM=5.3;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:86,25:5:5:0:0,0,5,0 chr16 9152718 . GC G 18.4763 PASS INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;BQBZ=-1;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,TAG,GTT;BBEG=9152627;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=72;DPLX_CLIP=0;DPLX_NM=8.5;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:48,0:4:4:0:0,0,4,0 chr16 9152718 . GC G 57.4183 PASS INDEL;IDV=7;IMF=1;DP=7;VDB=6.71664e-05;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GTT,TAG;BBEG=9152626;BEND=9152991;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=17;DEPTH_NORM_REV=0;DPLX_ASXS=96;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=60;BULK_NM=1;NN=[0:169:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:110,23:7:7:0:0,0,7,0

sample 10 chr16 9152718 . GC G 31.4548 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,TGG,ATA;BBEG=9152627;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=84;DPLX_CLIP=0;DPLX_NM=6;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:93,32:5:5:0:0,0,5,0 chr16 9152718 . GC G 61.1452 PASS INDEL;IDV=11;IMF=0.916667;DP=12;VDB=2.26006e-08;SGB=-0.676189;RPBZ=0;MQBZ=0;BQBZ=-3.31662;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=1,0,11,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CTT,AAG;BBEG=9152626;BEND=9152991;DEPTH_FWD=5;DEPTH_REV=7;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=101;DPLX_CLIP=0;DPLX_NM=2;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:131,43:12:11:0:1,0,11,0 chr16 9152718 . GC G 51.2953 PASS INDEL;IDV=9;IMF=0.9;DP=10;VDB=1.02904e-05;SGB=-0.662043;RPBZ=-1.93649;MQBZ=0;BQBZ=-2.23607;SCBZ=-1.93649;MQ0F=0;AC=1;AN=1;DP4=1,0,9,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,TAA,GAG;BBEG=9152626;BEND=9152991;DEPTH_FWD=5;DEPTH_REV=5;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=78;DPLX_CLIP=0;DPLX_NM=5.2;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:78,0:10:9:0:1,0,9,0 chr16 9152718 . GC G 78.3164 PASS INDEL;IDV=10;IMF=0.909091;DP=11;VDB=6.99472e-07;SGB=-0.670168;RPBZ=0;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=1,0,10,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,TAG,AAA;BBEG=9152627;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=7;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=94;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:121,16:11:10:0:1,0,10,0 chr16 9152718 . GC G 27.5107 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00558876;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,GGG,TCA;BBEG=9152627;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=75;DPLX_CLIP=0;DPLX_NM=5.5;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:89,32:5:5:0:0,0,5,0 chr16 9152718 . GC G 97.4152 PASS INDEL;IDV=15;IMF=1;DP=15;VDB=8.40038e-10;SGB=-0.688148;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,15,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,CAG,CGA;BBEG=9152627;BEND=9152992;DEPTH_FWD=8;DEPTH_REV=7;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=3;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:127,0:15:15:0:0,0,15,0 chr16 9152718 . GC G 75.2638 PASS INDEL;IDV=9;IMF=0.9;DP=10;VDB=5.52906e-06;SGB=-0.662043;RPBZ=0;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=1,0,9,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GAT,TAA;BBEG=9152626;BEND=9152992;DEPTH_FWD=6;DEPTH_REV=4;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=61;BULK_NM=1;NN=[0:165:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:102,0:10:9:0:1,0,9,0

sample 32 chr16 9152718 . GC G 51.4182 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,AAT,AGT;BBEG=9152626;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=100;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:110,29:5:5:0:0,0,5,0 chr16 9152718 . GC G 60.4147 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00248765;SGB=-0.590765;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CAA,GGG;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=92;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:90,0:5:5:0:0,0,5,0 chr16 9152718 . GC G 40.4249 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.58114;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,AGA,TCT;BBEG=9152626;BEND=9152992;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=94;DPLX_CLIP=0;DPLX_NM=3.7;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:104,34:5:5:0:0,0,5,0 chr16 9152718 . GC G 77.4149 PASS INDEL;IDV=8;IMF=1;DP=8;VDB=1.33331e-05;SGB=-0.651104;BQBZ=-2;MQ0F=0;AC=1;AN=1;DP4=0,0,8,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GGT,TTA;BBEG=9152626;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=4;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=3.3;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:107,0:8:8:0:0,0,8,0 chr16 9152718 . GC G 68.4148 PASS INDEL;IDV=6;IMF=1;DP=6;VDB=0.000560625;SGB=-0.616816;BQBZ=-2.23607;MQ0F=0;AC=1;AN=1;DP4=0,0,6,0;MQ=60;QPOS=92;RB=chr16,9152626,9153566,TGT,TAG;BBEG=9152626;BEND=9153566;DEPTH_FWD=4;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=95;DPLX_CLIP=0;DPLX_NM=2.5;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:98,0:6:6:0:0,0,6,0 chr16 9152718 . GC G 72.4187 PASS INDEL;IDV=10;IMF=1;DP=10;VDB=6.99472e-07;SGB=-0.670168;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,10,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GCA,TAA;BBEG=9152626;BEND=9152992;DEPTH_FWD=7;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=91;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:121,19:10:10:0:0,0,10,0 chr16 9152718 . GC G 47.4146 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00248765;SGB=-0.590765;BQBZ=-2;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,GGT,AAT;BBEG=9152626;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=93;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:77,0:5:5:0:0,0,5,0 chr16 9152718 . GC G 41.4185 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.73205;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,CTT,CTG;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=92;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:84,13:5:5:0:0,0,5,0 chr16 9152718 . GC G 69.4148 PASS INDEL;IDV=7;IMF=1;DP=7;VDB=0.00011279;SGB=-0.636426;BQBZ=-1.87083;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=91;RB=chr16,9152627,9152992,GAC,TGA;BBEG=9152627;BEND=9152992;DEPTH_FWD=4;DEPTH_REV=3;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=81;DPLX_CLIP=0;DPLX_NM=5.8;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:99,0:7:7:0:0,0,7,0 chr16 9152718 . GC G 60.4147 PASS INDEL;IDV=6;IMF=1;DP=6;VDB=0.000411984;SGB=-0.616816;BQBZ=-1.58114;MQ0F=0;AC=1;AN=1;DP4=0,0,6,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,AGG,CCC;BBEG=9152627;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=4;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=91;DPLX_CLIP=0;DPLX_NM=4.3;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:90,0:6:6:0:0,0,6,0 chr16 9152718 . GC G 60.218 PASS INDEL;IDV=10;IMF=0.909091;DP=11;VDB=6.99472e-07;SGB=-0.670168;RPBZ=0;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0;MQ0F=0;AC=1;AN=1;DP4=1,0,10,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,GAG,AGG;BBEG=9152627;BEND=9152991;DEPTH_FWD=7;DEPTH_REV=4;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=94;DPLX_CLIP=0;DPLX_NM=3.5;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:145,58:11:10:0:1,0,10,0 chr16 9152718 . GC G 62.4147 PASS INDEL;IDV=5;IMF=1;DP=5;VDB=0.00187095;SGB=-0.590765;BQBZ=-1.49071;MQ0F=0;AC=1;AN=1;DP4=0,0,5,0;MQ=60;QPOS=91;RB=chr16,9152627,9152991,TCG,GTC;BBEG=9152627;BEND=9152991;DEPTH_FWD=3;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=103;DPLX_CLIP=0;DPLX_NM=1.7;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:92,0:5:5:0:0,0,5,0 chr16 9152718 . GCC G 70.4187 PASS INDEL;IDV=4;IMF=1;DP=4;VDB=0.0058656;SGB=-0.556411;BQBZ=-1.73205;MQ0F=0;AC=1;AN=1;DP4=0,0,4,0;MQ=60;QPOS=92;RB=chr16,9152626,9152992,CCA,GTA;BBEG=9152626;BEND=9152992;DEPTH_FWD=2;DEPTH_REV=2;DEPTH_NORM_FWD=19;DEPTH_NORM_REV=0;DPLX_ASXS=98;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=63;BULK_NM=1;NN=[0:215:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:115,15:4:4:0:0,0,4,0

sample 22 chr16 9152718 . GC G 51.4183 PASS INDEL;IDV=7;IMF=1;DP=7;VDB=9.50611e-05;SGB=-0.636426;MQSBZ=0;BQBZ=0;MQ0F=0;AC=1;AN=1;DP4=0,0,7,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,GTT,ACG;BBEG=9152626;BEND=9152991;DEPTH_FWD=2;DEPTH_REV=5;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=91;DPLX_CLIP=0;DPLX_NM=4;BULK_ASXS=61;BULK_NM=1;NN=[0:157:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:104,23:7:7:0:0,0,7,0 chr16 9152718 . GCC G 95.3043 PASS INDEL;IDV=13;IMF=0.928571;DP=14;VDB=3.3258e-08;SGB=-0.683931;RPBZ=0.27735;MQBZ=0;MQSBZ=0;BQBZ=0;SCBZ=0.27735;MQ0F=0;AC=1;AN=1;DP4=1,0,13,0;MQ=60;QPOS=92;RB=chr16,9152626,9152991,ATA,ATC;BBEG=9152626;BEND=9152991;DEPTH_FWD=4;DEPTH_REV=10;DEPTH_NORM_FWD=16;DEPTH_NORM_REV=0;DPLX_ASXS=98;DPLX_CLIP=0;DPLX_NM=2.7;BULK_ASXS=61;BULK_NM=1;NN=[0:157:0];SEQ=ATAAAGATGCCCCCCCCC GT:PL:DP:DV:SP:DP4 1:208,86:14:13:0:1,0,13,0

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gevro commented 8 months ago

Yes, these used restriction enzyme fragmentation. We also made mouse noise masks, but these sites got through.

So it sounds like there is no systematic filter to help with these other than to manually find them and add them to the noise mask?

fa8sanger commented 8 months ago

So it sounds like there is no systematic filter to help with these other than to manually find them and add them to the noise mask?

Indel realignment, which is computationally expensive, and the noise mask, right.

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cancerit / NanoSeq

QC for indels #80