Hi guys,

I have a collection of 5kb human sequences that I want to compare their differences, particularly if base changes result in nonsynonymous changes. I know there are tools such as VEP, ANNOVAR, etc that annotates changes, but they all seem to require a VCF first.

What is the simplest way to generate a VCF of their differences. I figure since there is no reference sequence for this region, I did a multiple sequence alignment. Perhaps I can make a consensus sequence and use that as reference for variant calling?

Since what I have are just Fasta sequences I am not sure how to further proceed. I did try jvarkit tool, biostar94573 from a previous thread, but it does not seem to work for me.

Thanks!