Mutation calling directly from FASTQ files
1
0
Entering edit mode
8 months ago
graeme.thorn ▴ 50

I have a list of variants that I would like to check exist in some cell-free DNA extracted from plasma. These variants arise from DNA extracted from clinical tissue, and tumour-derived variants may appear at very low levels in the cell-free DNA extracted from the same patients. The sequencing is such (using UMIs and consensus deduplication) that one or two variant-containing fragments post-deduplication would potentially be enough to confirm the presence of ctDNA in the plasma.

I have tried running the GATK pipeline and the samtools mpileup/mutscan pipeline, and both are excellent in extracting variants with allelic fractions close to 50%, but these are likely derived from genomic cfDNA rather than circulating tumour DNA.

Is there a bioinformatic method sensitive enough to detect the one or two DNA molecules containing the variant?

DNA-Seq variant calling • 335 views
ADD COMMENT
1
Entering edit mode

If you just need to call low frequency variants, why does it have to be directly from FASTQ files?

ADD REPLY
0
Entering edit mode

I was looking for a method sensitive enough for a single read that covers the variant

ADD REPLY
0
Entering edit mode

There are many variant callers that allow you to set thresholds (one nice overview). If you just want to check for any mismatches, you can use pileups directly. It's not necessary to add the extra caveats to deal with FASTQs directly.

ADD REPLY
1
Entering edit mode
8 months ago

What you seem to be looking for is called "alignment-free" variant calling (e.g. https://www.nature.com/articles/s41598-017-02487-5 or https://academic.oup.com/bioinformatics/article/34/10/1659/4657072).

However, I'm not sure if that is going to solve your problem. Alternatively, you could look at somatic variant caller, e.g. Mutect2. Those don't expect a 0.5 allelic ratio.

ADD COMMENT

Login before adding your answer.

Traffic: 1275 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6