Hi All,
Say you want to amplify 75 small-ish (250-400 bp) loci from a population sample of, say, 15 individuals. So just over 350kb total you want sequenced by MiSeq with 2 x 250.
Question 1: Is there any particular reason adaptors and barcodes can't be ligated to the amplicons as they would be in genome sequencing?
Question 2: If they can be ligated as such, is there any reason to expect that they would (or would not) efficiently ligate to primer dimers in non-purified PCRs?
Basically, I'm asking if you could just amplify a bunch of loci for a sample of individuals and if the PCRs look good (i.e., no huge primer dimer problems), pool them and sequence them. The hope being that the differences in locus-to-locus (and individual-to-individual) amplicon concentration do not result in huge cluster differences and that the coverage overkill compensates anyway.
Don't worry, I haven't actually tried this... yet.
P.S. The motivation for this is what I perceive to be a great difficulty in getting accurate quantitation/normalization of 75 x 16 = 1200 samples without robotics. Also, I have seen firsthand some poor recovery with Ampure XP beads, not to mention quite a lot of sample-to-sample variation in concentration after PCR clean-up.
Sorry in advance for what may be blinding naivete.
- Adam
Say you want to amplify 75 small-ish (250-400 bp) loci from a population sample of, say, 15 individuals. So just over 350kb total you want sequenced by MiSeq with 2 x 250.
Question 1: Is there any particular reason adaptors and barcodes can't be ligated to the amplicons as they would be in genome sequencing?
Question 2: If they can be ligated as such, is there any reason to expect that they would (or would not) efficiently ligate to primer dimers in non-purified PCRs?
Basically, I'm asking if you could just amplify a bunch of loci for a sample of individuals and if the PCRs look good (i.e., no huge primer dimer problems), pool them and sequence them. The hope being that the differences in locus-to-locus (and individual-to-individual) amplicon concentration do not result in huge cluster differences and that the coverage overkill compensates anyway.
Don't worry, I haven't actually tried this... yet.
P.S. The motivation for this is what I perceive to be a great difficulty in getting accurate quantitation/normalization of 75 x 16 = 1200 samples without robotics. Also, I have seen firsthand some poor recovery with Ampure XP beads, not to mention quite a lot of sample-to-sample variation in concentration after PCR clean-up.
Sorry in advance for what may be blinding naivete.
- Adam
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