FLX+ and amplicons

[suefo]

Good afternoon all. I understand there are people out there now using the FLX+ with amplicons (which is now fully supported by 454 / Roche). We are contemplating an upgrade from FLX to FLX+ but would appreciate some feedback.

1. How big are your read lengths?
2. Do you calculate an error rate and have you tracked between FLX and FLX+
3. I'm looking over the protocols now, but are there any significant difference in the sequencing methods between FLX and FLX+?
4. We are looking at 16S. What regions are people using on the FLX+ if also sequencing 16S?
5. General thoughts or opinions about FLX+ / amplicons?

Thank you. You can either reply to this thread or email me directly: Sue Foltin@ [email protected]

[JackieBadger]

My thoughts on amplicon studies are to avoid 454 and go straight to MiSeq.
Illumina data is cheaper and less error prone.

[suefo]

We have a MiSeq... but we need to read across larger regions.

[Dynamac]

Sue,

We recently upgraded to FLX+ but haven't carried out any runs other than test runs with control beads. I can tell you that the upgrade went smoothly, was performed on site in about 3 days, including the test run. There is a change in the hardware, but the system is backward compatible in case you still have old sequencing kits that you want to utilize. The protocols change a little, but not significantly. Also, there is an additional maintenance step required every 3 months (replacement of some tubing). And of course, the runs and processing take longer.

We likely will do 16S eventually, but the first thing we're going try is a shotgun library, prepared by restriction enzyme digestion and genome reduction. We will be doing 700-800 bp COI amplicons soon as well. I can keep you informed if you'd like.

Barry