454 FLX+ with amplicons - anyone tried it?

[rglover]

I guess the title says it all - has anyone tried sequencing amplicons on an FLX+ yet? We're looking at trying it in the next month or two and I was wondering whether anyone else had tried it already and had any success.

[mohdsos]

well, I am waiting for the upgrade, I am so doing it. but the is a bump, ROCHE won't support that very run, as amplicons is not listed as FLX+ application, so in case that something went wrong, there will be no replacement or any sort of support. I guess you can do it anyway. tell me what your results will look like.


Thanks
Mo

[Abha]

We have done amplicons on the FLX plus with mixed results. Sequencing works very well on the amplicons containing only one type of amplicon (median readlengths of 600bps even though seem like there are more sequecing errors). However, if you pool amplicons of different size and different loci, it completely stuffs up (Lot more key pass and filter pass failures). Apparently due to high signal intensities. Since, Roche will not support it hard to get much information out from them. Not sure why this happens but would be great to get some feedback from any one who has been able to get it working.

[proteasome]

We've seen the same problem: when mixing amplicons, particularly of different sizes, FLX+ has significant issues. I would note that we routinely pool different sized amplicons on a GS-Jr and don't see any abnormalities in read length or distribution. In the FLX+ runs we've done the reads are either short or scant or both