Sequencing advice

[nkaushik]

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?

[bioinfosm]

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..

[NextGenSeq]

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