Hi, I have some SNPs with flanking sequences ~60 kb both upstream and downstream of a SNP. The flanking sequence looks some thing like this. ATTATGGCT[A/T]TTAGCTTA (this is just an example). My question is 1) I am making my own fasta file by using this information and converting aforementioned sequence as snp_c1 ATTATGGCTATTAGCTTA and doing blast. I am keeping ref allele only and in this case it is A. So is this a correct way of adding flanking sequence info to query or should I make a fasta file like this and do BLAST? snp_c1 ATTATGGCTATTAGCTTA (this is flanking sequences with ref allele) snp_c1 ATTATGGCTTTTAGCTTA (this is flanking sequence with alt allele)
2) Second thing whats the best way to filter blast hits? I am keeping only unique hits but I am confused about the optimal thresholds that I can apply on bit score and e-value? Is there any threshold to do filtering when it comes to SNP's?