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  • blastn help for primer alignment to custom db

    Hello!

    I am trying to find primer pairs that will amplify across 6 individuals of the same taxa. I have forward and reverse primers(<30bp each) for each individual that were generated by QDD, and am now trying to blast the primers from each individual to the microsatellite sequences(msat + flanking regions) from the other 5 (ex. primers from A blasted to msat sequences from B,C,D,E,F). The goal is to find which primers will align in all 6 individuals so that we can order the primers and test if they amplify in the lab.

    This is the blast command I'm using right now. I want the results to be only alignments that span 100% of the query sequence. Really, a global query:local database alignment.

    >>blastn -task blastn-short -qcov_hsp_perc 100 -perc_identity 100 -ungapped -num_threads 8 -outfmt 6 -query "query.fa" -db "subject.db" -out out.out

    Unfortunately, I'm really new to this and am not very knowledgable on the subject, but does it look like this command will give me the results I am looking for? Thanks for the help!

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