May I consult with you that if GATK pipeline can be used to call mutations for the cell-free circulating DNA sequencing data? If so, is there any difference or any points to pay attention to?
Thank you very much in advance!
Hi there, sorry for the very late response -- been busy with preparing a workshop.
I think that sounds reasonable; you'll want to run each sample through the GATK data pre-processing workflow, then do the calling with MuTect on the matched sample pairs. Exact details depend on which version of MuTect you use. We have a new beta version of MuTect2 coming out in a few days (see this presentation for details) but it's not been approved yet for production use. If you're doing clinical production work you should use the older MuTect which doesn't do indel calling and does require an additional "co-cleaning" step (indel realignment on tumor/normal samples together). That is described in the Cibulskis et al. paper.
This is not something we have tried ourselves so we can't give you any canned advice. I would expect you'd have to deal with a number of complications (related to coverage, purity etc) depending on what you are trying to learn from the cfDNA data. Can you give us a bit more context on your use case / experimental design?
Thank you for your response! I got a data set, including 30 patients with plasma cell free DNA and matched PBMC DNA as an internal control that reflects the genomic DNA from the patient. I need to call somatic mutations for these patients. I guess I can use bwa-picard-GATK realignment and recalibration pipeline and switch to MUTECT or other tools for variant calling. Am I right? Any input is very appreciated!
Yes, I did what you said. Thank you! It is good to know that MuTect2 can do both SNV and indel now.
FYI mutect2 is now available in the newly released version 3.5
I have cfDNA samples of 4 patients and we have taken two normal samples, now how can i call variants using Mutect2 if i dont have matching normal and patient pairs?
Please follow this doc for info on variant calling with mutect2 without matching normal and patient pairs.