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wdemos

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#1

Varscan v2.3.5 headers missing

08-29-2013, 05:03 AM

I am running varscan to call variants on a single file set. I have run it twice now and the header files are missing on the output vcf files. All other samples I analyzed with varscan this week (11), have the header files. What might cause the header to not be included?

I used bwa to align my illumina fastq files.
I am using samtools v 1.1.19 for the mpileup.
My command line argument:
samtools mpileup -q 1 -C50 -DSf /ref/human_g1k_v37.fasta /myData/S12_sorted.bam | java -jar VarScan.v2.3.5.jar mpileup2snp --min-coverage 8 --min-reads2 2 --min-var-freq 0.01 --min-avg-qual 15 --p-value 0.01 --strand-filter 0 --output-vcf 1 --variants 0 > /myData/S12_snp.vcf

Here is the header on my .bam file:
-bash-3.2$ samtools view -H /myData/S12_sorted.bam
@SQ SN:1 LN:249250621
@SQ SN:2 LN:243199373
@SQ SN:3 LN:198022430
@SQ SN:4 LN:191154276
@SQ SN:5 LN:180915260
@SQ SN:6 LN:171115067
@SQ SN:7 LN:159138663
@SQ SN:8 LN:146364022
@SQ SN:9 LN:141213431
@SQ SN:10 LN:135534747
@SQ SN:11 LN:135006516
@SQ SN:12 LN:133851895
@SQ SN:13 LN:115169878
@SQ SN:14 LN:107349540
@SQ SN:15 LN:102531392
@SQ SN:16 LN:90354753
@SQ SN:17 LN:81195210
@SQ SN:18 LN:78077248
@SQ SN:19 LN:59128983
@SQ SN:20 LN:63025520
@SQ SN:21 LN:48129895
@SQ SN:22 LN:51304566
@SQ SN:X LN:155270560
@SQ SN:Y LN:59373566
@SQ SN:MT LN:16569
@SQ SN:GL000207.1 LN:4262
@SQ SN:GL000226.1 LN:15008
@SQ SN:GL000229.1 LN:19913
@SQ SN:GL000231.1 LN:27386
@SQ SN:GL000210.1 LN:27682
@SQ SN:GL000239.1 LN:33824
@SQ SN:GL000235.1 LN:34474
@SQ SN:GL000201.1 LN:36148
@SQ SN:GL000247.1 LN:36422
@SQ SN:GL000245.1 LN:36651
@SQ SN:GL000197.1 LN:37175
@SQ SN:GL000203.1 LN:37498
@SQ SN:GL000246.1 LN:38154
@SQ SN:GL000249.1 LN:38502
@SQ SN:GL000196.1 LN:38914
@SQ SN:GL000248.1 LN:39786
@SQ SN:GL000244.1 LN:39929
@SQ SN:GL000238.1 LN:39939
@SQ SN:GL000202.1 LN:40103
@SQ SN:GL000234.1 LN:40531
@SQ SN:GL000232.1 LN:40652
@SQ SN:GL000206.1 LN:41001
@SQ SN:GL000240.1 LN:41933
@SQ SN:GL000236.1 LN:41934
@SQ SN:GL000241.1 LN:42152
@SQ SN:GL000243.1 LN:43341
@SQ SN:GL000242.1 LN:43523
@SQ SN:GL000230.1 LN:43691
@SQ SN:GL000237.1 LN:45867
@SQ SN:GL000233.1 LN:45941
@SQ SN:GL000204.1 LN:81310
@SQ SN:GL000198.1 LN:90085
@SQ SN:GL000208.1 LN:92689
@SQ SN:GL000191.1 LN:106433
@SQ SN:GL000227.1 LN:128374
@SQ SN:GL000228.1 LN:129120
@SQ SN:GL000214.1 LN:137718
@SQ SN:GL000221.1 LN:155397
@SQ SN:GL000209.1 LN:159169
@SQ SN:GL000218.1 LN:161147
@SQ SN:GL000220.1 LN:161802
@SQ SN:GL000213.1 LN:164239
@SQ SN:GL000211.1 LN:166566
@SQ SN:GL000199.1 LN:169874
@SQ SN:GL000217.1 LN:172149
@SQ SN:GL000216.1 LN:172294
@SQ SN:GL000215.1 LN:172545
@SQ SN:GL000205.1 LN:174588
@SQ SN:GL000219.1 LN:179198
@SQ SN:GL000224.1 LN:179693
@SQ SN:GL000223.1 LN:180455
@SQ SN:GL000195.1 LN:182896
@SQ SN:GL000212.1 LN:186858
@SQ SN:GL000222.1 LN:186861
@SQ SN:GL000200.1 LN:187035
@SQ SN:GL000193.1 LN:189789
@SQ SN:GL000194.1 LN:191469
@SQ SN:GL000225.1 LN:211173
@SQ SN:GL000192.1 LN:547496
@RG ID:work SM:S12 PL:Illumina PU:S12
@PG ID:bwa PN:bwa VN:0.5.9-r16

Thanks for any advice.
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dkoboldt

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#2

09-03-2013, 08:11 AM

Hello, and thanks for posting. Do you mean that the VCF header lines are missing? That's strange behavior and I'm happy to help you investigate.

Would you mind letting me know what your output file (/myData/S12_snp.vcf) looked like?
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wdemos

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#3

09-06-2013, 05:05 AM

Thanks for your help. Here is what the first 10 lines look like:
1 10061 . T G . PASS ADP=268;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:33:270:268:250:11:4.12%:4.385E-4:33:29:210:40:4:7
1 10067 . T G . PASS ADP=270;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:24:274:270:260:8:2.96%:3.7047E-3:33:31:207:53:7:1
1 10079 . T G . PASS ADP=266;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:33:268:266:254:11:4.14%:4.3923E-4:32:34:196:58:11:0
1 10083 . C G . PASS ADP=284;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:71:284:284:261:23:8.1%:7.496E-8:34:32:188:73:23:0
1 10097 . T G . PASS ADP=236;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:55:240:236:213:18:7.66%:2.7036E-6:30:29:149:64:17:1
1 10108 . C T . PASS ADP=188;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:49:189:188:170:16:8.51%:1.0897E-5:32:33:111:59:4:12
1 10109 . A T . PASS ADP=183;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:145:184:183:139:44:24.04%:2.9982E-15:31:30:81:58:29:15
1 10114 . T G . PASS ADP=172;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:37:173:172:155:12:7.14%:1.9895E-4:31:25:88:67:5:7
1 10147 . C A . PASS ADP=49;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:39:49:49:37:12:24.49%:1.1358E-4:33:27:12:25:11:1
1 10177 . A C . PASS ADP=39;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:33:39:39:20:10:25.64%:3.9851E-4:27:32:4:16:4:6
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wdemos

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#4

09-06-2013, 05:06 AM

My apologies I forgot to disable the smilies in text:
1 10061 . T G . PASS ADP=268;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:33:270:268:250:11:4.12%:4.385E-4:33:29:210:40:4:7
1 10067 . T G . PASS ADP=270;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:24:274:270:260:8:2.96%:3.7047E-3:33:31:207:53:7:1
1 10079 . T G . PASS ADP=266;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:33:268:266:254:11:4.14%:4.3923E-4:32:34:196:58:11:0
1 10083 . C G . PASS ADP=284;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:71:284:284:261:23:8.1%:7.496E-8:34:32:188:73:23:0
1 10097 . T G . PASS ADP=236;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:55:240:236:213:18:7.66%:2.7036E-6:30:29:149:64:17:1
1 10108 . C T . PASS ADP=188;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:49:189:188:170:16:8.51%:1.0897E-5:32:33:111:59:4:12
1 10109 . A T . PASS ADP=183;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:145:184:183:139:44:24.04%:2.9982E-15:31:30:81:58:29:15
1 10114 . T G . PASS ADP=172;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:37:173:172:155:12:7.14%:1.9895E-4:31:25:88:67:5:7
1 10147 . C A . PASS ADP=49;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:39:49:49:37:12:24.49%:1.1358E-4:33:27:12:25:11:1
1 10177 . A C . PASS ADP=39;WT=0;HET=1;HOM=0;NC=0 GT:GQ:SDPP:RD:AD:FREQ:PVAL:RBQ:ABQ:RDF:RDR:ADF:ADR 0/1:33:39:39:20:10:25.64%:3.9851E-4:27:32:4:16:4:6
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wdemos

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#5

09-16-2013, 12:39 PM

Should I provide a different type of data in order to trouble shoot? Thanks
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wimufi

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#6

09-24-2013, 10:26 AM

Hi, I am having a similar problem with VarScan 2.3.6. Is there any workaround for this? I can provide sample input/output if you need it.
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gaoch

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#7

10-22-2013, 04:32 AM

Anyone have fixed this problem? I also meet this issue, VCF header is missing in some, while is right in others. I don't know why could this happen.
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wdemos

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#8

10-22-2013, 06:38 AM

I have not resolved this issue yet and am working on it today. I will let you know if I find anything.
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wdemos

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#9

06-05-2014, 10:18 AM

Has anyone else had this issue. I am testing this software again. I've run the same exome sample with three different aligners. All of the bam files have headers. Only 2 of the 6 analyses I've run were output with headers. I'm still stumped as to why this is occurring. Thanks.
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d3d1y

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#10

04-27-2015, 08:27 PM

Yep. Me too. Exactly the same situation: illumina fastq to bam via bwa; samtools mpileup to vcf via varscan --output-vcf. Resulting vcf has no header.
Latest Ubuntu, latest samtools, latest varscan as of March 2015.
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wdemos

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#11

04-28-2015, 08:05 AM

I still have not resolved this issue. Please keep us posted if you find a solution. Thank you
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d3d1y

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#12

04-28-2015, 08:29 PM

The problem is that --output-vcf 1 doesn't work for pileup2snp, it only works for mpileup2snp. Lack of documentation for this flag, plus lack of error checking for command line args, makes it hard to figure this out.
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