SEQanswers

Go Back   SEQanswers > Bioinformatics > Bioinformatics



Similar Threads
Thread Thread Starter Forum Replies Last Post
Samtools mpileup - Call somatic mutations from a pair of samples - detail steps ? swapnil2188 General 2 09-23-2013 04:49 PM
CASIM: Variants, Filtering SNP Calls casim UK - Cambridge 0 04-18-2013 12:29 PM
VarScan2 somatic output (genotype call) desmo Bioinformatics 1 12-27-2012 07:49 AM
varscan2: filters mrfox Bioinformatics 1 10-09-2012 08:40 AM
Homozygous Ref calls and no calls ashkot Bioinformatics 1 08-09-2012 10:01 AM

Reply
 
Thread Tools
Old 06-06-2013, 12:54 AM   #1
ElenaN
Junior Member
 
Location: Moscow, Russia

Join Date: Nov 2012
Posts: 8
Default Filtering steps for VarScan2 somatic and germline calls

What is the proper filtering pipeline for somatic mutation detection using VarScan2, with tumor/normal paired samples?
My understanding of the steps:
0. VarScan2 somatic to make calls
Then filter:
1. VarScan2 processSomatic to separte calls Germline/Somatic/LOH and call some "high confidence"
2. VarScan2 somaticFilter on the Somatic.hc file to do further filtering.
- as I understand, this is where filtering for false positives aroudn indels take place
- it also seems to look at p-values and such - is it not redundant with the previous step?
3. fpfilter.pl on the resulting data for further filtering?
(in my hands this step seems to hang indefinitely without doing anything, though. not sure why)

My questions are:
- is this a correct order of steps, or are some of the 3 filtering steps redundant or obsolete?
- what are the recommended parameter settings for bam-read-count, especially mapping quality q? Should it be 1, as is recommended for mpileup, or higher?

Now, if I want to detect germline mutations in the same sample, can I just use the "Germline" (and I guess LOH) calls from VarScan?
Dan Koboldt seems to think so, as long as one uses the normal bamcount in step 3. Should "VarScan2 filter" be used in step 2? Or should a separate germline mutation caller be used for this purpose?

Thank you,
Elena
ElenaN is offline   Reply With Quote
Old 06-28-2013, 12:56 PM   #2
dkoboldt
Member
 
Location: St. Louis

Join Date: Mar 2009
Posts: 62
Default

Elena,

You could probably just perform steps 1 and 3 and get a good high-confidence set of somatic mutation calls.... the somaticFilter command is more simplistic than the filter false positives.

If you want the germline calls, combine Germline.hc and LOH.hc files, and then run that through the false positive filter with the NORMAL bam.

As for bam-readcount, I'd recommend at least mapping quality of 1 and base quality of 15.
dkoboldt is offline   Reply With Quote
Old 07-03-2013, 01:43 AM   #3
ElenaN
Junior Member
 
Location: Moscow, Russia

Join Date: Nov 2012
Posts: 8
Default

Thanks, Dan!
(BTW, I just posted another VarScan question as a separate thread http://seqanswers.com/forums/showthr...364#post109364 - it'd be really great if you could answer it as well.)
ElenaN is offline   Reply With Quote
Old 11-08-2013, 11:12 AM   #4
zhixiang
Junior Member
 
Location: uchicago

Join Date: Oct 2012
Posts: 2
Default

Quote:
Originally Posted by dkoboldt View Post
Elena,

You could probably just perform steps 1 and 3 and get a good high-confidence set of somatic mutation calls.... the somaticFilter command is more simplistic than the filter false positives.

If you want the germline calls, combine Germline.hc and LOH.hc files, and then run that through the false positive filter with the NORMAL bam.

As for bam-readcount, I'd recommend at least mapping quality of 1 and base quality of 15.
Hi dkoboldt,

Can filterfp.pl be used to filter the somatic indels?

Can I combine the indel vcf and snp vcf from the output of varscan somatic, and then filter the vcf file using step 1 and step 3? Does this works for both snp and indel in the vcf file?
zhixiang is offline   Reply With Quote
Old 08-27-2014, 04:57 AM   #5
thek71
Junior Member
 
Location: Freiburg

Join Date: Mar 2011
Posts: 4
Default

Hi all,
I have a similar question regarding the germline mutations. I have several tumor-normal samples for which I have used the somatic command and I want to check several genes for germline mutations. From the publication is stated that hc variants are the ones with at least 10% allele frequency in normal and tumor samples.
This varinat is in the hc file:
chr1 565088 C T 0 37 100% T 4 19 82.61% T Germline 1.0 0.018159073897484498 2 2 10 9 0 0 22 15
while this one
chr1 741267 T C 33 38 53.52% Y 30 22 42.31% Y Germline 5.540944545408712E-23 0.9211526256316275 2 28 3 19 10 23 5
33
is not. The numbers of reads and the percentages seem to be ok, but I fail to understand why the variants are categorized differently.
Any help would be highly appreciated.

Thank you in advance
thek71 is offline   Reply With Quote
Reply

Tags
germline mutations, somatic mutations, varscan

Thread Tools

Posting Rules
You may not post new threads
You may not post replies
You may not post attachments
You may not edit your posts

BB code is On
Smilies are On
[IMG] code is On
HTML code is Off




All times are GMT -8. The time now is 08:03 AM.


Powered by vBulletin® Version 3.8.9
Copyright ©2000 - 2018, vBulletin Solutions, Inc.
Single Sign On provided by vBSSO