Anyone else having Lib-A GS Jr problems recently?

[proteasome]

Recently we've been having problems related to the quality of our Lib-A sequencing on the Jr. Very few passed filter reads, mixed-like flowgrams when our enrichment is correct, or problems with the short filter. We are not having similar problems with Lib-L runs.

We've done over 200 runs on the Jr with good success, so I know our lab technique is not an issue. Wondering if it might be a reagent problem. Has anyone else been having similar problems with recently ordered Lib-A kits?

Simon

[mccarya]

I noticed your thread while searching for posts for a similar issue we're having.

We have a FLX system, but have had multiple runs (at least 5) with almost no passed filter, and very short reads even though pre emPCR Agilent traces show no dimer. I was wondering if you have resolved the issue? If so, would you mind sharing what you did?

We have changed lots, sample source and primer sets with the same results, and are at a bit of a loss as to what to do next. I'm attaching the most recent read length histogram, in case anyone is seeing similar problems. Thanks!

[proteasome]

We have not been able to figure out what the problem is yet. For us, bad runs seem to come in batches so we figured it was a problem with one of our lots.

Some things to consider, based on our previous troubleshooting: Have you tried sequencing beads that have worked before? What do your filterpass reads look like? is there a repetitive sequence motif that keeps coming up in your sequences?

If you keep having problems you should contact GS Support and they might be able to help you. Another way to troubleshoot would be to have your libraries sent to a sequencing center or another lab with a Jr or FLX and have them run them, in order to prove to Roche that it's not your libraries that are causing the problem.

[pmiguel]

Yeah, amplicons do not seem to work as well as they did a few months ago for us.

Try reprocessing the run using the non-amplicon pipeline. The normal one.
See:

http://pathogenomics.bham.ac.uk/blog/2011/05/curious-results-from-454-amplicon-processing/

for a good overview.

I have a suspicion that Roche is tinkering with their emPCR kit formulation to get them to work well with the new gsflx+ chemistry and normal Titianium stuff is suffering unexpectedly. But that is complete speculation on my part.

About the short amplicon issue -- Roche makes it devilishly hard to figure out whether your reads are short because they hit the primer on the other side of the insert (short amplicon) or the read just sputtered out early for some other reason. So, I was thinking it would be a good idea to run libraries on an Agilent RNA (pico) chip after heating them (95 oC 2 minutes followed by snap chill on ice prior to loading) to see if there are small single stranded amplicons annealed to the larger amplicons. But I have not gotten around to it yet.

Also, if your amplicons are >500 bp, there would probably no down side to using the GS-FLX+ emPCR protocol, instead of the Titanium one. Or at least giving it a shot. It is mainly just a different thermal cycler program.

Oh, I should add that we took our off rig processing pipeline up to v2.6, but left the software on the 454 at v2.5. I have been meaning to do the upgrade on the 454 as well. So, that leaves the possibility that there is some minor and unnoticed incompatibility between v2.5 image analysis and v2.6 signal processing. Or that v2.6 processing in general has been de-tuned in some way that is resulting in sub-par amplicon results.
--
Phillip

[proteasome]

I second the idea of reprocessing with a non-amplicon pipeline and turning many of the quality filters off. We have successfully uncovered problems (such as incompatibilities between our amplicon and the sequencing adapters) by using this method and interrogating the reads manually.

As for longer amplicons, while the FLX+ thermocycling conditions couldn't hurt, I've successfully sequenced many long amplicons (some over 1 kb) with the standard conditions and had good recovery and sequencing.