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  • Sequencing advice

    I am planning to sequence 60kb gene using ovelapping long range PCR, I have designed primers and amplified whole gene. I am running into problem now because I am amplifying mixtures of sequences of another gene that is highly homologous (93%) to my gene.

    When I sequence mixture of amplified population, will I will be able to characetrize SNPs specific to my gene by aligning to reference genome?

  • #2
    If it is so homologus, you are bound to see mis-alignment. It might be a good idea to check out mappability features for your genes of interest. Perhaps a longer read application would be more suited for you..
    --
    bioinfosm

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    • #3
      Always BLAT your primer sequences prior to having them synthesized. If they map to more than a single region in the genome redesign them.

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