FLX+ Library Amplification Issue

[Dynamac]

We are having problems getting our long-fragment shotgun library to amplify during emPCR. Titration using 3 different cpb ratios (12, 18, and 24) yields the same percentage enrichment (3-4%, which doesn't even give us enough beads for a 2-region PTP). A test aqueous phase PCR using Lib-L primers A and B also gives very poor amplification.

This is a library prepared by restriction enzyme digestion, genome reduction, gel purification and size selection, and finally adaptor ligation. This process has been reported to work in the literature, albeit with the FLX chemistry.

I'm really perplexed about the non-dependence of cpb on % enrichment. This might be a clue about what's going on. Anyone have any ideas about what is happening?

I haven't sequenced these beads. I had a look at the ssDNA left in the first melt supernatant on a RNA Nano Chip and saw lots of short fragments and only a smithen of 1000 base product.

Thanks.

Barry

[ajthomas]

I wouldn't sequence the beads if I were running it. The fact that you saw lots of short fragments indicates that even if there is sequence on those beads the sequence is no good. The information you give (poor performance of the library in liquid-phase PCR coupled with the equal enrichment of all three titrations) suggests that you may have some contamination and the beads you got were mostly from the contaminant rather than the library. There may be other explanations, but that's my first guess.

[Dynamac]

Thanks, Phillip. I didn't sequence them.

I don't know how much faith I have in the aqueous PCR result since there isn't a Roche protocol (that I know of) for testing FLX+ shotgun libraries. I was flying by the seat of my pants. I ran a positive control along with it, but that positive control was a Lib-L 16S amplicon, which may not be appropriate. At any rate, I'm going to clean the library again and Roche is going to send me different lots of reagents to try. Could be bad oil.

[Dynamac]

Oops. You aren't Phillip, are you. Sorry about that, I'll just say AJ.

[ajthomas]

No worries. I've been called worse.