How to design primers when I only know the position of the mutation given in the VCF file?

Question

[jpxaviersantos90]

Hi,

I am facing quite a challenge, which I believe is very easy to solve, but I am not being able to do so. In my work I analyzed exome sequencing from individuals with a given disorder and searched for single nucleotide variants (SNVs) targeting specific genes of interest. Now, I will perform Sanger sequencing in some of the samples, to check if the mutation is really there. My analysis was done using VCF files which, as you probably are aware, give the coordinates in which the SNV occurs. For the sequencing I have to design primers. For example, I know that 1 individual has a mutation in gene A, The SNV is in chromosome X, position xxxxxxx. My doubt is not (of course) how I design the primers, but where do I get the reference sequence so that I can design them, knowing that I want my forward and reverse primers to go from 100bp before and after the SNV. There are so many tools I can access, just by going to NCBI, that I get somewhat confused on the right way to do this...

Thanks in advance and sorry for my poor English.

Answer 1

[jared.andrews07]

Take your SNV position, add/subtract 100 or so from it, and plug those coordinates into UCSC's DNA tool to get the sequence in FASTA format. Lots of other high-throughput methods to pull the reference sequence for a given position as well - bedtools, pyfaidx, samtools faidx come to mind among others.