Mpileup/BCFtools pipeline not picking up indels (suggestions please)

[cam.jack]

Hi Everyone. I've googled long and hard for this, as well as trying every parameter that I can think of, but my indel detection pipeline is only picking up SNPs.

I know this because I introduced 104 copies of an artificially created insertion and 104 copies of an artificial deletion, which are present in my SAM file.

The relevant section of my pipeline is as follows (just a bash script):

$TOOLS/samtools mpileup -E -C50 -g -e15 -o30 -F0.005 -P ILLUMINA -uf $INDEX $SAM.sorted.bam > $SAM.bcf

$TOOLS/bcftools view -vcg -i0.001 $SAM.bcf > $SAM.vcf

cat $SAM.vcf | grep "INDEL" > $SAM.indels.vcf

Unfortunately there are no entries marked as INDEL in the resulting VCF file.

Any suggestions as to what I might be doing wrong would be most welcome!

Thanks for reading.

Cameron

[oiiio]

Do you mind posting or messaging me a section of the VCF output?

[oiiio]

At any rate, you could try running the with only the -uf and -g parameters in mpileup. And if all else fails you could follow the exact command lines of the instructions on http://samtools.sourceforge.net/mpileup.shtml

I literally just used the command lines in the instructions with the latest version of samtools, and got both SNPs and indels in my VCF file.

[cam.jack]

Can't see how to private message you sorry. Here is a section my VCF for chromosome 4. I'll give your suggestion a crack now. Would still love know what's going on. The manuals only ever hint at what works best...

4       146901815       .       G       A       42      .       DP=23;AF1=0.5;CI95=0.5,0.5;DP4=11,7,4,1;MQ=47;FQ=45;PV4=0.62,1,1.3e-08,1        GT:PL:GQ        0/1:72,0,255:75
4       146901885       .       C       T       134     .       DP=13;AF1=0.5;CI95=0.5,0.5;DP4=3,3,4,2;MQ=45;FQ=118;PV4=1,1,1,1 GT:PL:GQ        0/1:164,0,145:99
4       146901944       .       C       A       15.1    .       DP=18;AF1=0.5;CI95=0.5,0.5;DP4=3,10,3,1;MQ=45;FQ=18.1;PV4=0.099,1,3.5e-12,1     GT:PL:GQ        0/1:45,0,245:48
4       146901989       .       T       A       8.64    .       DP=20;AF1=0.5;CI95=0.5,0.5;DP4=5,9,4,1;MQ=44;FQ=11.3;PV4=0.14,0.091,5.1e-11,1   GT:PL:GQ        0/1:38,0,255:40
4       146901997       .       T       C       20      .       DP=18;AF1=0.5;CI95=0.5,0.5;DP4=5,8,4,1;MQ=44;FQ=23;PV4=0.29,1,1.1e-10,1 GT:PL:GQ        0/1:50,0,255:53
4       146902038       .       G       C       55      .       DP=15;AF1=0.5;CI95=0.5,0.5;DP4=5,5,2,3;MQ=44;FQ=58;PV4=1,0.095,9.7e-06,1        GT:PL:GQ        0/1:85,0,237:88
4       146902109       .       C       G       58      .       DP=21;AF1=0.5;CI95=0.5,0.5;DP4=7,4,9,0;MQ=40;FQ=61;PV4=0.094,0.021,0.0052,0.19  GT:PL:GQ        0/1:88,0,227:91
4       146902112       .       G       A       63      .       DP=23;AF1=0.5;CI95=0.5,0.5;DP4=7,4,10,0;MQ=38;FQ=66;PV4=0.09,0.039,0.0041,0.14  GT:PL:GQ        0/1:93,0,222:96
4       146902171       .       T       C       162     .       DP=26;AF1=0.5;CI95=0.5,0.5;DP4=9,4,10,3;MQ=40;FQ=165;PV4=1,0.095,0.0065,0.07    GT:PL:GQ        0/1:192,0,247:99
4       146902302       .       G       A       35      .       DP=22;AF1=0.5;CI95=0.5,0.5;DP4=5,13,0,4;MQ=44;FQ=38;PV4=0.54,1,1,1      GT:PL:GQ        0/1:65,0,255:68
4       146902307       .       C       T       136     .       DP=20;AF1=0.5;CI95=0.5,0.5;DP4=1,9,1,8;MQ=44;FQ=139;PV4=1,0.41,0.37,1   GT:PL:GQ        0/1:166,0,179:99
4       146902377       .       T       G       94      .       DP=9;AF1=0.5;CI95=0.5,0.5;DP4=0,3,0,6;MQ=44;FQ=36;PV4=1,1,0.018,1       GT:PL:GQ        0/1:124,0,63:66
4       146915129       .       C       A       5.46    .       DP=2;AF1=0.5002;CI95=0.5,0.5;DP4=1,0,1,0;MQ=47;FQ=3.86;PV4=1,1,1,1      GT:PL:GQ        0/1:34,0,30:32
4       146938077       .       C       T       4.77    .       DP=2;AF1=0.5;CI95=0.5,0.5;DP4=0,1,1,0;MQ=47;FQ=5.09;PV4=1,1,1,1 GT:PL:GQ        0/1:33,0,34:32
4       146938236       .       C       T       10.4    .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,0,1;MQ=44;FQ=-30  GT:PL:GQ        1/1:40,3,0:5
4       146948997       .       C       T       10.4    .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,1,0;MQ=44;FQ=-30  GT:PL:GQ        1/1:40,3,0:5
4       146949118       .       A       C       7.8     .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,0,1;MQ=44;FQ=-30  GT:PL:GQ        1/1:37,3,0:4
4       146950985       .       C       G       17.8    .       DP=3;AF1=1;CI95=0.5,1;DP4=0,0,2,0;MQ=29;FQ=-33  GT:PL:GQ        1/1:49,6,0:10
4       146951252       .       A       C       13.2    .       DP=39;AF1=0.5;CI95=0.5,0.5;DP4=4,25,0,9;MQ=44;FQ=16.1;PV4=0.55,1,1e-12,1        GT:PL:GQ        0/1:43,0,255:46
4       146951379       .       A       G       29      .       DP=10;AF1=0.5;CI95=0.5,0.5;DP4=0,7,0,3;MQ=32;FQ=32;PV4=1,1,1,1  GT:PL:GQ        0/1:59,0,102:62
4       146953212       .       G       A       10.4    .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,0,1;MQ=44;FQ=-30  GT:PL:GQ        1/1:40,3,0:5
4       146955437       .       T       G       225     .       DP=89;AF1=0.5;CI95=0.5,0.5;DP4=11,11,30,34;MQ=36;FQ=222;PV4=0.81,0.14,4.3e-08,1 GT:PL:GQ        0/1:255,0,250:99
4       146956645       .       C       T       53.1    .       DP=7;AF1=1;CI95=0.5,1;DP4=1,0,0,6;MQ=25;FQ=-28;PV4=0.14,1,1,1   GT:PL:GQ        1/1:82,1,0:4
4       146956678       .       T       C       65.1    .       DP=6;AF1=1;CI95=0.5,1;DP4=0,0,0,6;MQ=25;FQ=-45  GT:PL:GQ        1/1:98,18,0:33
4       146957306       .       A       G       73      .       DP=21;AF1=0.5;CI95=0.5,0.5;DP4=0,12,0,7;MQ=42;FQ=76;PV4=1,0.35,0.0081,0.36      GT:PL:GQ        0/1:103,0,166:99
4       146958185       .       G       T       10.4    .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,1,0;MQ=44;FQ=-30  GT:PL:GQ        1/1:40,3,0:5
4       146975399       .       A       T       5.29    .       DP=2;AF1=1;CI95=0.5,1;DP4=0,0,0,2;MQ=19;FQ=-33  GT:PL:GQ        1/1:35,6,0:6
4       146977925       .       A       G       9.53    .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,0,1;MQ=44;FQ=-30  GT:PL:GQ        1/1:39,3,0:5
4       146980527       .       C       T       9.53    .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,1,0;MQ=44;FQ=-30  GT:PL:GQ        1/1:39,3,0:5
4       146985507       .       A       G       10.4    .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,1,0;MQ=44;FQ=-30  GT:PL:GQ        1/1:40,3,0:5
4       146987884       .       G       A       38      .       DP=14;AF1=0.5;CI95=0.5,0.5;DP4=6,3,5,0;MQ=40;FQ=41;PV4=0.26,0.46,0.0014,1       GT:PL:GQ        0/1:68,0,209:71
4       146987922       .       T       C       7.8     .       DP=16;AF1=0.5;CI95=0.5,0.5;DP4=7,4,3,1;MQ=41;FQ=10.4;PV4=1,0.03,0.00016,0.2     GT:PL:GQ        0/1:37,0,242:39
4       146987923       .       G       C       7.8     .       DP=16;AF1=0.5;CI95=0.5,0.5;DP4=7,4,3,1;MQ=41;FQ=10.4;PV4=1,0.017,0.00016,0.21   GT:PL:GQ        0/1:37,0,242:39
4       146987945       .       G       C       93      .       DP=21;AF1=0.5;CI95=0.5,0.5;DP4=9,3,7,1;MQ=43;FQ=96;PV4=0.62,0.11,0.00073,0.47   GT:PL:GQ        0/1:123,0,242:99
4       146988379       .       G       A       7.8     .       DP=1;AF1=1;CI95=0.5,1;DP4=0,0,1,0;MQ=37;FQ=-30  GT:PL:GQ        1/1:37,3,0:4
4       146989474       .       T       C       6.2     .       DP=29;AF1=0.4999;CI95=0.5,0.5;DP4=2,12,7,6;MQ=28;FQ=8.65;PV4=0.046,1,1.8e-10,1  GT:PL:GQ        0/1:35,0,210:36
4       146989642       .       T       C       61      .       DP=10;AF1=0.5;CI95=0.5,0.5;DP4=0,5,0,4;MQ=31;FQ=33;PV4=1,1,1,1  GT:PL:GQ        0/1:91,0,60:63

[oiiio]

Any luck with the indels showing up on only the minimum options?

[swbarnes2]

If you could show us a few entries in the sam file that cross your artificial indels, that might help. The pileup file too (mpileup without the -g) around the area where you think the indel is.

[cam.jack]

Very strange but it seems that it was the piping from one process to another that broke it!

I've now taken every process call and separated it out on to its own line and now it works.

Thanks for your help everyone. I'll now try out the parameters I was using to see if they improve things.

[narain]

I am having hard time with samtools as well. It misses so many SNPs and InDels which is clearly visible by visualizing the BAM file in IGV.