Titanium Runs

[pr0t3us]

Hi to everybody,

we have a 454-Titanium. We made some runs, but we have never obtained good results.
With 454-GS-FLX, we have never had problems; major of runs was like expected. With Titanium we obtain poor runs (less reads than expected, strange read length distribution, less raw weels then expected) .

Has someone the same problems ?

[Drizzt]

Have you just started experiencing these problems? Are you having problems enriching beads as well? What percentage of raw wells are you seeing? Example, how many beads are you loading and how many are registering as raw wells?
I would talk with your Roche/454 rep and see if they have any information.

Hi to everybody,

we have a 454-Titanium. We made some runs, but we have never obtained good results.
With 454-GS-FLX, we have never had problems; major of runs was like expected. With Titanium we obtain poor runs (less reads than expected, strange read length distribution, less raw weels then expected) .

Has someone the same problems ?

[SeqWhiskey]

We're having the same problem. Raw wells are lower by an order of magnitude than what they should be, read length is much lower than it should be it just tapers off, very few quality reads. Our FLX runs have been excellent though and we just ran an FLX after the most recent Ti failure... We're contacting our rep, tech support and everyone else we can get ahold of but... so far Ti has not delivered. at all.

[SeqWhiskey]

We just got the new emPCR kits with the mysterious "additive", so hopefully this helps. It is supposed to improve bead quality and we can now start using beads with up to 20% enrichment, much different than the previous 8% recommendation.... When I talk to our Roche tech I'll let you know if we find anything useful.

Best, Matt

[Drizzt]

We just got the new emPCR kits with the mysterious "additive", so hopefully this helps. It is supposed to improve bead quality and we can now start using beads with up to 20% enrichment, much different than the previous 8% recommendation.... When I talk to our Roche tech I'll let you know if we find anything useful.

Best, Matt

The "additive" should increase your output and read length. It is interesting that some kits have had this problem and others haven't been affected within the same lot numbers. I have not used the additive but have heard good things from other labs. I would be interested in hearing your results.


D

[genomeseeker]

If your raw wells are lower than expected check these things:
- equipment used for bead counting after enrichment; new settings required for Ti's smaller beads.
- enrichment procedure; maybe you're carrying over a large amount null beads to your sequencing run.

[boss_hoss]

I agree with genomeseeker - if the raw wells are low, this indicates a problem with the final enriched bead count going into the sequencing reaction.

If your read length distribution is strange, consider changing your cpb going into emPCR. This may be an artifact of mixed signals.

Both these problems could be linked - if an improper count was found after enrichment, enrichment % would be miscalculated.