[boerm]

preincubation of enrichment beads with enrichment primer

So, did it help? We are still doing it. Current work-around is to underload the plate and simply expect to have to do two runs to get the number of reads that we routinely got up until Dec 2011. Been nearly a full year of this, now.

Dear HMorrison,

Yes, preincubation of the enrichment beads with the enrichment primer as you suggested worked in our LV lib-L. The problem now is that the enrichments is now suddenly 40%, while from the SV (done without preincubation step) with the same cpb (1,5) gave 5% enrichment.

[lilyhu]

We also suffered from low enrichment problem, mostly below 2%. The template that we are using is amplicon with fusion primer (with adaptor and barcode). We have checked the template with bioanalyzer and KAPA qPCR first. Then we used 1.7 cpb in the LV Lib-L emPCR. The enrichment is lower than 2% that we suspected the cpb not optimize, emPCR failed or the problem of the template? Then we went for SV using various cpb from 0.25 to 17, again the enrichment was <2% while the positive control (from previous work fine library) is OK but cpb increased from 1.7 to 14.6.

I am working on library instead of amplicon. I thought cpb is a relative number depending how you test you library concentration.

[exon]

We also suffered from low enrichment problem, mostly below 2%. The template that we are using is amplicon with fusion primer (with adaptor and barcode). We have checked the template with bioanalyzer and KAPA qPCR first. Then we used 1.7 cpb in the LV Lib-L emPCR. The enrichment is lower than 2% that we suspected the cpb not optimize, emPCR failed or the problem of the template? Then we went for SV using various cpb from 0.25 to 17, again the enrichment was <2% while the positive control (from previous work fine library) is OK but cpb increased from 1.7 to 14.6.

[lilyhu]

Spread a drop of emulsion on the cover of 96-well plate or other plastic and look under inverted microscope with a phase contrast. One can easily see if the emulsion is stable, that is droplets are not merging.

Thanks a lot!

[yaximik]

Spread a drop of emulsion on the cover of 96-well plate or other plastic and look under inverted microscope with a phase contrast. One can easily see if the emulsion is stable, that is droplets are not merging.

[lilyhu]

Emulsion oil lot likely will affect stability of the emulsion. For example, I found that I get much better distribution of beads in droplets when I use different mixing conditions (I use IKA Turrax). The protocoll calls for 5 min/4000 rpm premixing and 5 min/2000 rpm after adding beads, but I found that a lot of droplets still contain multiple beads. 3000rpm/10 min or 2X 3000rpm/5min produced the majority of droplets with single beads, so this is what I am using. Yet this did not eliminate the downstream problems.

How do you know the distribution of beads? thanks.

[yaximik]

Emulsion oil lot likely will affect stability of the emulsion. For example, I found that I get much better distribution of beads in droplets when I use different mixing conditions (I use IKA Turrax). The protocoll calls for 5 min/4000 rpm premixing and 5 min/2000 rpm after adding beads, but I found that a lot of droplets still contain multiple beads. 3000rpm/10 min or 2X 3000rpm/5min produced the majority of droplets with single beads, so this is what I am using. Yet this did not eliminate the downstream problems.

[lilyhu]

I have found the sticky beads was associated different lot of emulsion oil.

[yaximik]

I have exactly the same problems with GS Jr sine last spring - sticky beads, low numbers of raw wells, a lot of wells without the key, and a lot of short reads. As the number of control wells stays about the same and control reads are good, this got to be a emPCR issue. Sticky beads problem was easiest to resolve - amount of enrichment beads was empirically determined to be sufficient for less than 20% enrichment. That is, since beads are about 0.8 um in size, it takes a lot of them to hold strongly a much larger capture bead (~30 um) on the magnet. To many "positive" capture beads result in weak interaction making washes very frustrating. Positive means beads that have a complement to the enrichment oligo, which may come just from primer dimers or heterodimers. These are too short for sequencing, lack the key - that is where empty wells, short reads and no-key reads are likely coming from.

I used larger amounts of capture beads - any 0.8 um streptavidin magnetic beads work fine - and the problem of sticky beads gone. But all other downstream problems remained.

I think it is not the problem with the enrichment kit, but with the emPCR reagent kit. Somehow it favors formation of primer dimers/heterodimers screwing up everything downstream.

[HMorrison]

We will try preincubating the enrichment beads with the enrichment primer, thanks for the suggestion.

So, did it help? We are still doing it. Current work-around is to underload the plate and simply expect to have to do two runs to get the number of reads that we routinely got up until Dec 2011. Been nearly a full year of this, now.

[boerm]

emPCR lot numbers

In our last 5 failed runs we used 3 different lot number of emPCR reagents, but the lot numbers of the enrichment beads were all the same, eg. 93866020 (emPCR recovery reagents)

[lilyhu]

We have the same problem with Lib_L emPCR. Does anybody think this problem associated with different lot number of emPCR kit?

[boerm]

sticky enrichment beads

We also encounter the same problem of sticky enrichment beads, we see the same results, short reads and high percentage dot and mixed reads, probably due to the null beads being present on the PTP plate.

We use the REMe unit for enrichment, and since this is a black box, you have no idea what happens, so it took a while before we decided to do the enrichment manually again, and yes the beads stick together and needed 25 washes instead of the programmed 11 washes on the REMe system. The recovery was much lower in the LV as calculated from the titration which was still done on the REMe system.

We have the upgrade on the 454 and use the plus kits, so here the problems are probably more extreme.

We will try preincubating the enrichment beads with the enrichment primer, thanks for the suggestion.

[HMorrison]

pre-annealing enrichment primer

Hi,

This is the protocol (developed by Sharon Grim, who uses this forum but doesn't post often):

Combine 12.5 ul enrichment primer, 80 ul washed enrichment beads, 900 ul enhancing fluid. Anneal on the rotator for 5 min. This can be done in parallel with the melt step. After, wash 3x on the magnetic rack with 500-1000 ul enhancing fluid and resuspend in 80 ul volume again.

Problem is that it uses up enrichment primer over what is supplied in the kits.

[Aniki]

Dear HMorrison,

thank you for your help. Can you please explain a little about how to pre-anneal the primers? I would love to know that as this way I can at least rule out of the issues.

Thank you very much.