Zippy, did you use rapid libraries for your shotgun? If so, were you sure you did the denaturation step in the emPCR set-up? A low enrichment, plus such a high proportion of mixed reads would point to non-denatured rapid library in the emPCR. You would have dsDNA going into the emPCR with each strand being in opposite directions, hence all reads would come out as being mixed.
There is a theoretical limit for how many beads you can get back from emPCR, so it could be that you're well beyond that limit. Reducing the input may not result in the same proportional drop in enriched beads. For our 16s studies, we've found that using the Bioanlyser sizing, plus a Qubit/Picogreen quantification gives us hightly reproducible results on our FLX. We pretty much get 10% enrichment +/- 2% every time with 0.5cpb.
If in any doubt, use qPCR (such as the Kapa kit) but be aware that we've found different library types require different emPCR input.
There is a theoretical limit for how many beads you can get back from emPCR, so it could be that you're well beyond that limit. Reducing the input may not result in the same proportional drop in enriched beads. For our 16s studies, we've found that using the Bioanlyser sizing, plus a Qubit/Picogreen quantification gives us hightly reproducible results on our FLX. We pretty much get 10% enrichment +/- 2% every time with 0.5cpb.
If in any doubt, use qPCR (such as the Kapa kit) but be aware that we've found different library types require different emPCR input.
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