GS Junior - bead issues.

[KorNor]

Anyone else using a GS Junior?
I've set up ours and done a few runs - only moderately successful (user incompetence, ha!)

I am getting many more mixed beads than anticipated, even after quanting and using the calculator on the my454 site.

My bead counter always shows a level above 2mil at end stage.

Emulsions are not broken, etc etc

Anyone else had any problems like this with this platform? Any hints?

[Baseless]

Hello KorNor,

I can only speak from experience from the FLX, but there mixed beads result from too much DNA fragments per beads during the emPCR (and this is probably the situation in the entire workflow i fear the most).
I just took a look at the emPCR protocol for the Junior, it clearly states that if you have a bead count above 2min (this equals a recovery of more than 20%) you should not continue.
Since they claim your optimum window is between 500kBeads and 2mBeads, the window looks very familiar from the FLX (5-20%).

Empty or mixed beads are a very sensitive matter and even if someone needs data fast, never make compromises here, this can fail entire runs.

I suggest that you a) requantify your library and retry and/or b) cross check what I just said with customer support.

If you have access to a bead counter, you could determine the exact recovery rate and win an insight how much too much it actually was. But be aware that below 2% and above ~35 % these counts are also no longer trustworthy.

[KorNor]

Hi Baseless,

Yes, I understand that; the first time was during training run, and despite knowing we had over 2 mil beads, we pushed on, just to get the practice of setting up the run.
The next time, I reduced the template DNA by 1/3, still over 2 mil beads (I didn't put a seq run on with that). Repeated the emPCR again, using fresh library, again over 2 mil beads (again, I didn't put the seq run on).

I'll talk to tech support again next week.

[Palecomic]

Hi KorNor,

Just doing my training on newly installed GS Junior at the moment, we had exactly the same problem. More than double the upper limit of the range of beads in the bead counter. After interrogating the trainer, it seems that they've removed the titration step that's in the FLX protocol and specified 2 molecules per bead as ideal (in this case for 16S amplicon sequencing), but it looks like it's probably not good in this case.

Will see whether it improves next time, probably going to go with 0.5 and see whether the enrichment is more acceptable.

[KorNor]

Hi Palecomic,
Thanks for your reply; after speaking with tech support, I too have dropped the 2 molecules per bead in favour of a 0.6 - 0.8:1 ratio and that has been working well (if a little inconsistent). I think once you've done a few dozen runs, you'll probably have a better feel for it, but 2:1 is certainly much too high.
Otherwise, she's a good little breadmaker!

[Palecomic]

That's really good to know, thanks. I might suggest to them that in the next update to the manual they change that example.

Whereabouts are you? I'm not sure that there are that many Juniors about at the moment, I've just started a postdoc at Imperial.

Cheers

Mike

[plnagy]

Anybody using Agilent Sureselect routinely for capture for sequencing by the 454JR ?

[James Haile]

Splitting GS Junior Emulsion Oil

Has anyone tried splitting the GS Junior emulsion oil? i.e. adding the A beads (and Live A mix) to half the volume of oil, and the B beads (and Live B mix) to the other half? This way, if you don't need your sequences read in both directions, you could use two different bead to template ratios. Cheers!

[Palecomic]

plnagy: we haven't tried that system.

James: we haven't tried that either - I'm still getting to grips with the minutiae of the method, what would be the advantage of having both A and B beads in the same mix?

Do you mean you could set up A beads with 0.6 molecules per bead, B beads with 2 molecules and then capture them separately, sequencing whichever one had the level of enrichment that you wanted? It would avoid the issue of losing an emPCR kit every now and then to the wrong ratio.

I've only used the Lib-L kit so far, is separating the beads efficient and straight-forward?

[Zippy]

Hi KorNor et al.,

We've actually had a problem going the other way: despite our bead recovery being JUST at the 500,000 mark (i.e. barely recovering enough beads), we lost more than 98% of our reads to the mixed filter on two shotgun runs on different organisms. Emulsions aren't broken, and the sequence that does manage to pass the filter is good quality and maps well to our reference (suggesting that there isn't anything inherently wrong in our sample preps? Your thoughts? One of the library preps was set up under Roche supervision, the other exactly following the protocol...so unless their Junior library prep protocol has a whole in it?).

Control beads produce good sequence, and running Roche's canned E. coli library works fine (so there are no problems with our emuls-PCR technique or sequencing set-up...). We've tried reducing our input library by 1/3, but that has also reduced the amount of beads we recover to below the 500,000 magic limit. It is like we are getting overenrichment of beads, without overenriching! Tech support is thinking there might be a sample-specific problem in the emuls-PCR: anyone got any ideas? We are fresh out of them, and not looking forward to being told just to "run it again".

Any help would be super-greatly appreciated...

[Jacky76]

Hello

In my experience, you should get less enriched beads if you decreased the concentration (cpb). But if you are getting 2 mil. beads continuously, there must be some problem. Amplicons tends to be amplified during the emPCR more than shotgun. And if you are doing amplicon, you should check with PicoGreen. The assay is more accurate than Agilent calculation. And if you are doing shotgun, you don't have to leave the library and the standard longer than 6 hours after you finish the library prep. It is also recommended to measure mol/ul using fluorometer as soon as you can for using the calculator on my454.

[Soulbee]

hi, Ive had many cases with overenrichment with FLX and most were due to broken emulsions, detectable by settled beads in the wells after ePCR. However i did have one or two samples with continuous overenrichment and the tech support told me that it may be due to the nature of the sample. I would re-quantify my library and measure using picogreen or other similar measures like HS Qubit and calculate your molecules/ul. Maybe run a titration with 0.1cpb and 0.5cpb and make sure you wash as long as it takes until no white beads are coming off. Also, i have had a problem with some MV oils as it was resulting in broken emulsions ( detectable by settled beads in the wells after ePCR, whether or not there was a phase separation in the wells) which Roche replaced...

[yaximik]

My experience with GS Jr, perhaps, is the most illogical case of all. I am working with ancient DNA extracted from bone, not as old as Neanderthal, but quite old. Amounts of DNA extracted are at the detection limit of the Picogreen assay, so library quantification was likely unreliable. Based on this estimate using an about 50% of calculated recommended library volume, I got right amount of enriched beads, between 5e5 and 2e6. However, 98% did not pass the mixed bead filter. Next, I used 2 times less for emPCR. This time I got about 7% of valid reads, a tiny bit better. Yet, when I took 20 times less I got down only to 83% of lost reads. Make a little sense, huh?
Blaming library quantitation, I used SYBR Green qPCR with emPCR primers to requantitate the library.The estimate by SYBR Green was 100x higher than qPCR data! So it lloked like I was actually working with even less library than I thought, yet I was getting most of the reads lost to mixed bead filer. Makes even less sense , huh?
Anyway, I used this new concentration to load emPCR based on recommendd calculations. I got a way over 2e6 enriched beads. I reduced amount of library 5 fold, got a bit less packed enriched beads, but still a way over 2e6. Then I took 25 times less library, but I got even more packed enriched beads than the first time! This is as weird as it gets. I did not run any of these samples, but I am tempted to since the entire story makes no sense.
Tech support suggested that library denaturation may not be sufficient before loading. I do denturation step as recommended in the manual, except instead of letting thermocycler to chill to 4C, I stick the tube in ice to minimize annealing.
Another problem may be broken emulsions. In new emPCR kits, there is a yellow insert saying that oil formulation was changed and a top clear layer after PCR is normal, and does not indicate broken emulsion. However, that is exactly what shown in broken emulsion example in the manual, so it is very confusing now, since I always see this clear layer.
Does anyone experienced similar lack of logic?

[Palecomic]

Hi Yaximik,

With your emulsions, do you see a small clearing at the very bottom of the tubes?

We're doing amplicons and have carefully optimised our cpb ratio so that we get 5 % enrichment every time. We recently had an extra training session with some new staff, and the training kit included the newer oil along with yellow slip. The emulsions had a larger clear layer on the top as we were warned of, but also a very thin clear layer at the bottom. There was no sign of the classic banding and no pellet of beads that had dropped out of the emulsion. The emPCR gave us 20 % enrichment, much more enriched than expected, and the run failed due to over 50 % of the reads being mixed.

Another feature of the run was that of the reads that did make it past the filter, there were significant errors in the primer sequence (50 % again of the remaining reads). Roche confirmed that this was also the case with the control beads in the run, so perhaps it's worth checking this with the few reads that you do get?

I'm rerunning the same samples today with another kit, it does have the more turbid oil, but doesn't seem to include the yellow slip. I'll have to check, but I think it's a different lot number. Will let you know how it goes, but I've informed Roche and they are following up, so hopefully if this is another case of dodgy oil they'll track this through.

Over all, I've found that we've had a lot of run variability, and that if we sequence anything over 5 % enrichment, it's never a good run (20 % is definitely bad). Our pump has been replaced (as has the pump of one of the other guys with a Jr in the UK) and these are the first runs after that, making this trickier to troubleshoot, as we can't control all the other variables.

Good luck!

[Palecomic]

OK - to update, we have the same problem again. A very small volume of clearing at the bottom of the emPCR, no classic banding of the emulsion and this time it is possible to see a small pellet of beads.

I will be setting up the same library yet again with an older kit to double check that it's not other aspects of our work flow.

In the meantime if anyone comes across a similar problem to that detailed by myself or Yaximik it would be really useful if you could mention it here as well as to Roche. The lot number of oil I have a suspicion of is 93807320.

Thanks!