Hi all.

I am working on a matched tumor-normal somatic variant calling pipeline.
My pipeline is as below:

1. bwa mem alignment
2. sort and deduplicate with samtools and picard
3. Realignment with GATK
4. Base recalibration with GATK
5. Somatic mutation calling with Mutect / Varscan2
(For MuTect, I try both 1.1.7 and 2, with default setting. For VarScan2, I filter reads with mapping quality 20 and use processSomatic to pick out high-confidence calls)

Here are my questions:

1. When I filtered out the variants in introns/UTR/ncRNA, there are very little of intersection between Mutect/VarScan hit. The intersection between Mutect and Mutect2 is also very low. I am aware of the fact that the false positive rate is very high in current somatic mutation calling tools, but is there a way (a combination of parameter setting) that can filter out most of noises? (I know MuTect2 gives INDEL calling while old ones don't.)

2. I try to find a gold standard reference for whole exome sequencing. But what I've found so far are some articles using NA12878, simulating tumor mutation based on normal sample. Is there any reference I can use to evaluate my pipeline?

3. COLO829 is another candidate for me to use as reference. 
Since it is a genome sequencing sample, would it be ideal to use it as a reference standard, by using the exonic intervals?

I am still a novice in WES. Any reply would be greatly appreciated.

Thanks.





The DREAM challenge consortium has generated [synthetic cancer data sets][1] for benchmarking (whole genome, but you could easily filter to WES after alignment). Brad Chapman et al. at Blue Collar Bioinformatics have validated a lot of mutation-calling tools against this data (see [here][2]).


  [1]: https://www.synapse.org/#!Synapse:syn312572/wiki/62018
  [2]: http://bcb.io/2015/03/05/cancerval/