I want to sequence an amplicon library ~288 bp (including gene specific primers). I would like to do about 200 individuals and get back at least 300 reads/individual using dual indexing. Can I just make my own primers (order them, have them synthesized) with adaptor and barcodes attached and do single pcr like I used to with 454? I have found several bar code lists that are validated and seem to work. Its just a matter of knowing the Illumina MiSeq adaptor sequences right. Or, to maybe use the barcode/adaptor fusion primers again for something else, maybe do 2 pcrs, the first one with an overlap attached to my gene specific primer, the second with a complementary overlap on the fusion primers. It seems really simple, but all of the threads seem to include all kinds of confusing jargon I do not understand.
Has anyone else tried this--also, I do not see other threads that talk about per individual read count. I am studying a highly duplicated locus--maybe 20 variants per individual, so I need 200-500 reads/individual to make sure I can validate variants. It seems to me that a MiSeq should give back way more reads than that if I only load 200 samples.
Please bear with my ignorance!
Thanks
John
Has anyone else tried this--also, I do not see other threads that talk about per individual read count. I am studying a highly duplicated locus--maybe 20 variants per individual, so I need 200-500 reads/individual to make sure I can validate variants. It seems to me that a MiSeq should give back way more reads than that if I only load 200 samples.
Please bear with my ignorance!
Thanks
John
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