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I realize this is a familiar theme, but I thought I would chime in that our lab has tried (so far unsuccessfully) to do some amplicon sequencing on 100 bp libraries. The problems we've seen tend to fall into two categories:
1. Amplicon inserts shorter than expected after emPCR. Perhaps an issue where even small amounts of shorter contaminants can carry over from the earlier library-generating PCR, and are then preferentially amplified during emPCR. That there would be some bias isn't terribly surprising -- but we were surprised that what appeared to be good libraries by gel and/or bioanalyzer still showed this effect strongly.
2. Read lengths less than expected, given number of cycles. Even in cases where the amplicon length appears to be the correct length (that is, not surprisingly short or truncated), read lengths produced even by doing 120 cycles doesn't seem to be enough to produce full-length 100 bp reads.
Curious to hear other people's experiences here in tinkering with amplicons of various size and trying more or less # of cycles.
1. Amplicon inserts shorter than expected after emPCR. Perhaps an issue where even small amounts of shorter contaminants can carry over from the earlier library-generating PCR, and are then preferentially amplified during emPCR. That there would be some bias isn't terribly surprising -- but we were surprised that what appeared to be good libraries by gel and/or bioanalyzer still showed this effect strongly.
2. Read lengths less than expected, given number of cycles. Even in cases where the amplicon length appears to be the correct length (that is, not surprisingly short or truncated), read lengths produced even by doing 120 cycles doesn't seem to be enough to produce full-length 100 bp reads.
Curious to hear other people's experiences here in tinkering with amplicons of various size and trying more or less # of cycles.
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