How To Design Snp Flanking Primers For Non Model Species

Question

[upendrakumar.devisetty]

Hi, I would like to design some of the SNPs generated using Samtools mpileup pipeline between two genotypes of interest and validate them using PCR and Sanger sequencing. I am wondering is there an pre-existing tool to do that for non model species. What i have right now is vcf file and fasta sequence of the reference. Any help is appreciated. Thanks Upendra

Answer 1

[Michael]

If you have the reference sequence, there is not much special about designing primers for this case. You can use normal primer design tools like primer3, or eprimer3. If possible make sure that the SNP is well in the center of the PCR-product because the sequencing quality is normally best there. See also: primer

[upendrakumar.devisetty]

Thanks. But how do i extract flanking sequences surrounding SNPs to be able to design primers. I have looked at some threads on this forum but they all are intended for human or other model species and i work on non model species.

[Michael]

You know where your SNP is, and you have the sequence in a fasta file? Simply extract the sequence, there are many ways to do it, which you will easily find by searching the site.

[upendrakumar.devisetty]

So basically if i include 250bp on either side of the SNP position and extract the sequence based on those coordinates, i can design primers using that info. Right?

[Michael]

Sounds like a plan. Also, blast your primers against all your reference sequences to ensure uniqueness (using blast+ with -task blastn_short). Or simply use Primer blast http://www.ncbi.nlm.nih.gov/tools/primer-blast/ : under 'Primer Pair Specificity Checking Parameters' set option 'Database' to 'Custom' and upload reference genome. Under "PCR Template" upload your SNP flanks.