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  • cheaper options than Nextera

    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

  • #2
    My lab was part of this paper screening amplicons in large mutagenized populations, and the 2 step PCR was used: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4457992/

    A relevant quote about capacity and # of amplicons (25 amplicons from 18,000 fish)
    We amplified and directly sequenced the largest fragments possible using available NGS technology, without shearing or otherwise fragmenting the template. The MiSeq platform generates 25 million 250 bp paired-end sequencing reads (500 cycle version 2 reagent kit). We estimated, given this capability, that we could sequence 25 250 bp fragments in two directions from each of the ~18,000 haploid genomes in the library at sufficient coverage to detect multiple reads of a mutant allele that appears only once in a single 288-fish library pool. We chose 250 bp fragments rather than 500 bp fragments so that the MiSeq paired-end reads would be fully overlapping (see Sequence analysis section below).
    Providing nextRAD genotyping and PacBio sequencing services. http://snpsaurus.com

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