Hi,
we plan amplicon sequencing (cyt b) of cca 100 individuals (museum samples) in a 454 Junior run. I want to perform either gel cutting or Ampure purification prior to the sequencing. I just wonder if there is some reason why to purify each PCR separately or if it is ok to perform the purification on the pool of the amplicons. Possibly, the quantification of the individual PCRs (fluorometric - Qubit) is not really precise without the purification step (i.e. the amount of primer dimers etc. which are removed by the purification may vary between the samples and thus bias the concentration estimates?). Does anyone have experience with that?
Thank you very much,
dejsha
we plan amplicon sequencing (cyt b) of cca 100 individuals (museum samples) in a 454 Junior run. I want to perform either gel cutting or Ampure purification prior to the sequencing. I just wonder if there is some reason why to purify each PCR separately or if it is ok to perform the purification on the pool of the amplicons. Possibly, the quantification of the individual PCRs (fluorometric - Qubit) is not really precise without the purification step (i.e. the amount of primer dimers etc. which are removed by the purification may vary between the samples and thus bias the concentration estimates?). Does anyone have experience with that?
Thank you very much,
dejsha
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