cfDNA targeted sequencing - analysis pipeline
I have human DNA samples isolated from tissue and from plasma, which were targeted sequenced on Ion Torrent. I've read some papers and blogs about the data analysis pipeline, but it looks like there is no standard way to do it yet.
The steps to analyse cfDNA (mutation analysis):
- adapters trimming
- quality filtering (Q<20; length<40; remove N bases - are these filters right?)
- mapping to reference with BWA
- add UMI sequences (is this step necessary?)
- Base Recalibration
- calling (with HaplotypeCaller or MuTect2 ?)
- annotated with ANNOVAR
- filter to keep somatic variants (which filters are recommended?)
As I am a noobie to cfDNA analysis, I would really appreciate any help or suggestion.
Thank you very much in advance
I am going to move your post into our Community Discussions -> General Discussion topic, as the Somatic topic is for reporting bugs and issues with GATK.
You can read more about our forum guidelines and the topics here: Forum Guidelines.
I am not super familiar with cfDNA analysis so other users may be able to provide more insight. However, I would recommend following the steps of the Mutect2 pipeline for calling somatic variants. As for the pre-processing of the data in the first part of your proposed workflow, you can follow this workflow for data pre-processing for variant discovery using Mutect2. For filtering the variants called by Mutect2, you can check out the tool documentation for FilterMutectCalls which contains information about the available filters. I hope this was helpful, and there are also a few other forum posts regarding cfDNA which may be useful for reference.
I'm already checking the steps on those links. I was hoping for some tips on how to adjust that same pipeline to cfdna analysis.
Even so, your suggestions are much appreciated. Let's see if other users can give me more recommendations about this.
Thank you for looking into those links. Hopefully, other users will be able to provide some more insight on any adjustments to the pipeline that may be useful for cfDNA. In general, I would recommend that sticking to the Best Practices Workflows will likely yield the best results.
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