Multiplexing PCR for targeted sequencing

[Dessi]

Dear All,

Has anyone done multiplex PCR for targeted sequencing on MiSeq?

What software have you used to design the primers?

If My Illumina sequencing adapters are part of the target region primers, wouldn't that overhand sequence interfere with my PCR, especially in a multiplex reaction?

I don't know why I feel so nervous having a primer overhang.

Please, share your experience, thoughts, etc.

Thank you!

Dessi

[thermophile]

are the genes that you are interested in common targets (16s, ITS...) or are you wanting to multiplex many different amplicons together?

[Number6]

If it's multiple loci, we have a manuscript just published online in Horticultural Research describing "AmpSeq," which works particularly well for SNP genotyping multiple loci. It's Yang et al., Horticultural Research 3: 16002, 17 February 2016. It's simply Illumina's tailed-amplicon strategy, but does describe how the primers were designed.

[Dessi]

thank you.