Mutation frequency against reference fasta

Question

[Luca]

Hi everyone. I have some Atac-seq data and i wanted to know if it's possible and how to compare those reads against a reference genome. I was thinking of doing it manually 1) Align against the reference genome 2) Choose a gene 3) Extract the reads that fell into that gene 4) Extract the fasta of that gene 5) Align reads vs fasta 6) Evaluate (i dont know in which way ) the mutation rate.

Does that sounds good? Does anyone have some idea ? I know that statistically might not be the best but in our experiment we are very confident that the mutation in our samples should always be there and be the same, so even though the statistic wont be strong we were hoping to seeing that.

[Matthias Zepper]

Sadly, neither your data nor your analysis strategy is really convincing to me. ATAC-seq data typically contains a large fraction of reads from mitochondrial origin and is also highly skewed in coverage, which may impede proper variant calls.

If you wish to proceed nonetheless, first perform a a proper ATAC-seq analysis. Recover the reads from the cleaned and deduplicated *.bam files and then run rnavar on them. This might work or not. But in general, you should ideally use WGS for variant calls.

[Luca]

Thanks for your explanation, that's clear! I have another question, would it work if I have only several replicates but from the same sample and compare them against the reference genome instead of another sample?