emPCR

[MEJ]

In August we encountered a problem with the delivery of emPCR kits, the oil was kept in back order and could only be delivered after a month. In the kit part with the oil a yellow note is included: the formulation of the emulsion oil has been changed but this does not adversely affects the kit performance. Since we use these kits we get variable enrichment results and have to (re-)pcr more then we had to do before. The sequencing runs seem to be ok.
Does anyone recognize this?

[vlee2]

we have the same problem, though I don't think that the problem is in reformulated oil. Since we started to use emPCR kits with new oil (which is actually easier to work with: better washing and less clumping of beads, but sometimes with leaking cups, sometimes with black particles in the oil), we started to have much greater inconsistency between lot numbers of emPCR reagents and even withing same lot number of reagents. I just complained to Roche about this last Friday and waiting for response.

vlad

[jOn]

Hi MEJ - I'm pretty convinced it could be the oil - at least in my case - drop me a message and we'll swap LOT numbers.

jOn

[wildung]

Roche says the new oil should have a clear layer on top of the reaction after the run. That means I now find it impossible to tell if an emulsion is broken. With the old oil I tossed 3 or more wells from each plate due to broken emulsions. Since I can't discard them they purify and as you should suspect they will be highly enriched and contribute to mixed reads. I got better results with the old oil.

[MEJ]

Thanks you all for replying!
Our emPCR problems started with the new oil. I can't really tell what goes wrong since we sometimes get good results and somtimes it's crap even with the same lot numbers.
After emPCR the emulsions show more or less phase separation, only when we see a clear phase separation we find an enrichment that is over 50%. With the old oil we got nice titration series, with the kit nowaday's we (sometimes) have one or more titration points completely of.
The 'sometimes' bothers me, can it be that the oil is less stable? I can't prove this since the results are variable. Fact is that we more often have to redo or fill up emPCR's before we can load a PTP. Roche know's all this but doesn't recognise it.

[MissDNA]

Our low enrichment problem started with emPCR reagent (Lib-L) lot# 9792920. We are getting less than 5% enrichment for our LV preps, in some cases less than 2%. Before that lot we never experienced low enrichment. Many times we were able to fill up a whole PTP using only 1 emulsion cup. For the past week I have been exchange emails with Roche´s people. So far they were not able to come up with any conving explanation.
We set up our first emPCR with a new oil lot yesterday. We did use the same emPCR reagent lot, which finally ended today. I am curious to see what happens today.
For us the whole inconsistent data started with Phase C. Prior to that all our emPCRs were performed at 1 or 2 cpb. Now days our range is all over the place.
It is very frustrating!

Today´s result: 0,45% enrichment out of a 2 cup emPCR. Worse enrichment rate ever.

[Apollo704]

We are also seeing an increase in the enrichment % when using oil lots after 93805620. We were able to adjust for this by dropping our CPB by 60%, though we are seeing more variability than we have in the past.

[genes123]

Our experience with the new oil is similar. We had some very unusual enrichment and mix-dot values even when using libraries that had previously given good results. Also results were not consistent from day to day. After realizing that we were not observing broken emulsion anymore, we excluded systematically wells that were more prone to broken emulsion in the past (specific wells on the outside rows of the plates). Since then, our enrichments and mix-dot values have gone back to normal.

[genes123]

Also, Roche found our observations interesting and hinted that they might had seen this elsewhere, but they haven't yet acknowledged the problem. We had even tested different types of plates and sealers at their suggestion, but it had not solve the problem.

[MissDNA]

In our case I don´t think was the oil but something in the emPCR kit. Since we´ve started using a different lot things got better. In our lab we hardly ever observed broken emulsions.
One of the things support told me to do when I complained about low enrichment, was the I should to re-titrated the libraries using the new oil. They come up with all sorts of absurd suggestions but don´t admit there is a inconsistence from lot to lot. It is hard to work which such a variation.

[Soulbee]

We have also had much inconsistencies with our emulsion oils between SV oil and MV/LV. With the MV, roche ended up replacing my reagents because they could not solve my problem of high enrichment %, after suggesting lid/sealer changes upon others... Broken emulsions are hard to tell as the solution = white and the beads are also white. it helps to hold the plate up to a bright light and looking for settled pellet of white beads at the bottom of the wells which indicate broken wells, whether or not you see a phase separation or not.

[MissDNA]

Good thing about Roche is every time they cannot solve the problem, they replace reagents. Our low enrichment problems "miraculously" ended when lot# 93792920 ended. They never admitted it was a lot problem.
I don´t think there is a real good correlation inbetween SV and MV/LV.