Titration useless?

[Anthony.287]

I have a few questions about an ongoing project. This is using 2 sets of the HMP primers, which a specific customer has been using for about a year. Over the course of the past year, using both SV and MV kits, I’ve narrowed down the ideal c/b ratio for his libraries to 0.025-0.1, when quantified with qPCR. Now that his project is nearing its end, he’s ordering 2-region plates, and I’ve been using the LV kits. The problem is that I’m getting very low enrichment, using the same c/b ratio that worked well with both SV and MV (0.05, in this case). Last week, I had ~1% for one pool and ~3% for the other. I went ahead and ran those, and they yielded decent sequence, albeit not enough. Due to another upstream issue with that particular set of samples, I wasn’t surprised. I did another LV emPCR yesterday and today with his next order, and I haven’t counted the enriched beads because I can see that there are not nearly enough (much less than last week). Is there any reason you can think of that a c/b ratio would work well with an SV or MV kit, and very poorly or not at all with the LV? I am using an analog repeater pipette to dispense the emulsions, not an electronic one, so it shouldn't be that. I was thinking that it might have something to do with the volume of diluted sample added to the capture beads, but the volume that I added was within the range instructed. I know that I've seen other posts about titration values not being scalable, but why not? Other than that possibility, I’m scratching my head...

[yaximik]

0.05 cpb is already fairly low, one should be ok witn just 0.1. If you have to go much lower than that, I would think your lib quantitation is off. RUWhat kit do you use? I found that so far KAPA Biosystems SYBR kit was better as compared with my home-made SYBR assay, which I also found is quite dependent on enzyme/mastermix. The most sensitive was one from Takara, but KAPA one still beats this. Also, check if your emulsion is good just by looking under scope, it should have most of bubbles with one bead. I had to adjust mixing parameters from recommended to get that. Another subtle issue is emulsion stability during cycling, since evaporation can throw it off as well. Contrary to recommendations, I use sealing sheets from BioRad and quite firm tightening of the lid to ensure minimal evaporation effects.

[Anthony.287]

Yeah, I though 0.05 would be too low as well, but based on titrations of libraries prepared using these same primers, it’s real close to the sweet spot. And, if all goes well in emPCR, with an input of 0.05 cpb, 5% of the capture beads will have a molecule in the microreacter with them, so you shouldn’t get more than 5% enrichment. (Also, see my thread “qPCR instead of titration”. I posted a couple of papers there that someone else had posted; I’m being too lazy right now to repost them…) I’ve seen 16% enrichment with a library made with these same primers, and with a 0.05 cpb ratio, with the MV kit; I chalked this up to small fragments annealed to the amplicons of interest or primer dimers that I’d somehow missed. This is why I’m surprised that there is such a difference between SV, MV and LV, and I don’t have the time or money to sequence the same library with each kit and compare the results.
For what it’s worth, I’m using the KAPA kit for quantification. I like it so far! Now, if I could just get some consistent results…

[Rachael]

You may like to run the lot numbers of the reagents you are using past Roche when you see inconsistent results to rule out any possible reagent issues.

The emPCR breaking kits with lot numbers 93848420, 93852420 and 93859020 have all been flagged over the past few months by Roche as having issues.

[Anthony.287]

You may like to run the lot numbers of the reagents you are using past Roche when you see inconsistent results to rule out any possible reagent issues.

The emPCR breaking kits with lot numbers 93848420, 93852420 and 93859020 have all been flagged over the past few months by Roche as having issues.

Where did you hear that? I don't doubt it at all, but how do you know this?
PS - I was using lot #93848420....

I also have some more information about the scalability of the emPCR, which I'll try to put together this evening.

[Rachael]

Our lab was experiencing inconsistent emulsion results and contacted our local Roche Technical Product Specialist specifically querying specific emPCR breaking kit lot numbers we had used. We were e-mailed back regarding the above three lot numbers, two of which we had been using.

The problem we saw with 93848420 was the density of the enrichment beads was weaker than normal (in approximately 40% of the kits we had). If you still have this lot number in stock, open up a few of the boxes and compare the density of the beads. Less enrichment beads resulted in the inefficient capture of the enriched DNA capture beads and therefore a much lower % recovery.

93852420 we had problems with low % recovery (below 5%). If we used a different kit with the same cpb, the emulsion would work every time. I was informed that the problem with this lot number though does not cause emPCR failure but, and I quote, "Rather than observing emPCR failure, this particular lot number was associated with a low raw well number in analysed seq runs".

[MissDNA]

We have had very low enrichment in our lastest emPCR, we increased cpb and got even less enriched beads. I will definitely check the lots we have.

When you say breaking kit you are talking about bead recovery reagents, or these are reagents for GS Jr.?

[Rachael]

Yes, that's right MissDNA, the bead recovery reagents. Reference Number 05 641 870 001

[MissDNA]

Thanks, Rachel.
I checked our lots and we have 5 boxes of 93848420 and 17 of 93852420. The really low enrichment we had recently was with lot# 93837120, in which we have observed weaker density beads. Wonderfull!
Since these lots have been flagged, are they replacing it or anything?

[ajthomas]

You may like to run the lot numbers of the reagents you are using past Roche when you see inconsistent results to rule out any possible reagent issues.

The emPCR breaking kits with lot numbers 93848420, 93852420 and 93859020 have all been flagged over the past few months by Roche as having issues.

That's interesting. Two days ago I ran some amplicon samples I had cleaned up with lot #93859020 and had some odd results when cleaning it up. I cleaned up several groups of samples over three different days with that lot. The first day, I got high enrichment values, two were about 20% and one was about 35%. The second day, (different amplicon libraries, but equivalent amplicons, plus a repeat of the one that was 35% the first day) I used less DNA, but the enrichments were still fairly high at 15-22%. The third day, the enrichment values were better (again different libraries, but equivalent amplicons). On all three days, especially the third day, I had to wash A LOT--probably 12-15 times--to get them clean. At first it seemed almost all of the beads were bound to the enrichment beads and I got virtually no DNA beads off on the first round. I would get a few more each round, and they never seemed to stop coming off.

I was a little nervous about running the sequencing plate thinking that what I had were just junk. In the end, they sequenced okay, but not great. This was an 8-region plate, and I usually get ~150K-180K raw wells per region and about 50-60% pass filter rate for a yield of ~70K-85K reads/region. This time, there were many fewer raw wells, 80K-100K for samples cleaned up on day 3 and ~140K-150K for samples on days 1 and 2, and the pass filter rate was a little lower. Overall, I got ~30K-40K reads for the samples cleaned up on day 3 and ~50K for the samples from days 1 and 2.

I was wondering if there was a problem with the reagents somewhere along the line but I hadn't called GS Support yet. That's good to know there's a known problem with the emPCR enrichment reagents. That would explain the problems I had this time around.

ETA:I just looked through the reagents we have on hand, and almost all of them are of the affected lots.

[Rachael]

What you describe is exactly the same problems that we had with lot #93859020. We were performing a very high number of washes, up to 20 washes on one occasion, which takes a very long time! We also used a lot of extra reagents in the process trying to work out what was happening, as our % recoveries were very variable. It wasn't until we had 2 LV emulsions fail with that lot number, which had passed titrations the day before with a different kit that we finally questioned Roche about the emPCR kits.

Talk to GS support regarding your issue, we are in the process of getting all emPCR breaking kits replaced that we had in stock with the lot numbers in question.

That's interesting. Two days ago I ran some amplicon samples I had cleaned up with lot #93859020 and had some odd results when cleaning it up. I cleaned up several groups of samples over three different days with that lot. The first day, I got high enrichment values, two were about 20% and one was about 35%. The second day, (different amplicon libraries, but equivalent amplicons, plus a repeat of the one that was 35% the first day) I used less DNA, but the enrichments were still fairly high at 15-22%. The third day, the enrichment values were better (again different libraries, but equivalent amplicons). On all three days, especially the third day, I had to wash A LOT--probably 12-15 times--to get them clean. At first it seemed almost all of the beads were bound to the enrichment beads and I got virtually no DNA beads off on the first round. I would get a few more each round, and they never seemed to stop coming off.

I was a little nervous about running the sequencing plate thinking that what I had were just junk. In the end, they sequenced okay, but not great. This was an 8-region plate, and I usually get ~150K-180K raw wells per region and about 50-60% pass filter rate for a yield of ~70K-85K reads/region. This time, there were many fewer raw wells, 80K-100K for samples cleaned up on day 3 and ~140K-150K for samples on days 1 and 2, and the pass filter rate was a little lower. Overall, I got ~30K-40K reads for the samples cleaned up on day 3 and ~50K for the samples from days 1 and 2.

I was wondering if there was a problem with the reagents somewhere along the line but I hadn't called GS Support yet. That's good to know there's a known problem with the emPCR enrichment reagents. That would explain the problems I had this time around.

ETA:I just looked through the reagents we have on hand, and almost all of them are of the affected lots.

[Rachael]

I've uploaded a photo of the enrichment beads for lot #93848420. These are two brand new tubes taken out of the emPCR breaking kit box, you can immediately see the difference in density of the beads. We inspected all of out kits that were in stock of lot lot #93848420 and removed from use the kits which had a lower density of enrichment beads. Had no problems with emulsions with this lot number after that.

In regards to #93852420, apparently it is less than 10% of kits affected which is below whatever their recall threshold is.

Thanks, Rachel.
I checked our lots and we have 5 boxes of 93848420 and 17 of 93852420. The really low enrichment we had recently was with lot# 93837120, in which we have observed weaker density beads. Wonderfull!
Since these lots have been flagged, are they replacing it or anything?

[MissDNA]

Thanks again for the information and picture, Rachel. We are going to check our boxes today and see if we still have beads with the problem. If so, we will remove from use and ask Roche for replacement.

[Anthony.287]

Rachael - thanks for the picture! One question; which tube is which?

After contacting Roche, I figured out that the reagents weren't the problem. The problem was that I was using the LV kit, when all of the titrations had been done with the MV. Apparently, the ratio of aqueous : oil is very important when creating the emulsions, and the ratios aren't maintained across the 3 different sized kits. For SV, the ratio is ~0.85-0.87, MV ~0.94-0.96 and LV ~0.88-0.90. I had assumed that the ratios would be the same, or at least very close to the same, across all of the kits, but it sure doesn’t look like it. Please feel free to double-check my math, but I think it’s right.

I went back to using the MV kit with the previously determined ideal cpb, and all is well.

[MissDNA]

Do you do titrations with MV? We always do with SV. Is my understand that titration should be scalable to MV and LV. However, we know that is not the case.

What do you mean by those ratios?

Today we are setting up emPCR LV for XL+ sequencing and for one library we are using 36 cpb and the other 56 cpb :s