[Benh]

!!!

I see what you mean now. Sorry I've been reading the post with my Lib-L beer goggles

Well, I'm glad that at least your problem seem to be sample specific. Thanks for sharing your experience.

[ajthomas]

Yes you're right that you won't see the A adapter sequence, but what one should see at the 3' end is:
-- insert -- reverse amplification primer -- key -- B adapter sequence

You will only see the key and adapter sequence in the flowgram and it will be trimmed in the output sequence.

What I see is:
-- part of insert -- B adapter sequence

The adapter sequence is not trimmed in the output sequence because the software doesn't see the key.

In a normal short fragment sequence situation, you will still see the reverse amplification primer, key, and B adapter sequence; the only difference is that the insert sequence will be different and will be short. As far as the sequencing chemistry is concerned, those are regular amplicons.

What I'm seeing is different: there is no reverse amplification primer or key, and the sequence is correct up to the point where I see the B adapter sequence. It's not a primer dimer. The only way I can explain it is if the emPCR amplification primer is identical to the entire adapter sequence minus the key, and it is mispriming during the emPCR step.

As I said before, there is no trace of small fragments on the BioAnalyzer trace and this affects almost every read in the run. I've only seen it happen with 16S amplicons, and only 16S amplicons from one user. Other amplicons from the same lab work just fine, even in the same run.

[Benh]

Thanks for the reply ajthomas!

If I understand your case correctly, it is actually normal to see only one amplification primer (amp. B sequence) but not A sequence+key because of where sequencing starts:

5' -- amp. A sequence -- [sequencing primer site] -- key -- MID -- insert -- amp. B sequence -- 3'

If the read is long (say >400nt) the sequencing quality at the 3' end will be so bad that B sequence will be trimmed automatically. I suspect that sequence B is retained in this case because the read is short enough that the sequencing quality is still good enough. That's my theory anyways, I hope that make sense...

If that's the case, I think your case is similar to ours, that is, small fragments are more readily amplified during emPCR.


Anybody else with short read problem? Surely there must be more frustrated 454 users out there....

[ajthomas]

I have seen lots of short reads with 16S amplicons since the beginning of the year as well. What I've seen, however, is not primer dimers or trimming, but it appears as though the Lib-A B amplification primer is mispriming in the amplicon. If I look at the flowgrams of these runs I can clearly see the B primer sequence at the end of the flowgram, but the reverse primer and the key sequence are missing. The pass filter rate will be good but the sequences are all short, usually about 73 bp long. The Bioanalyzer trace shows no short molecules at all in these libraries.

This only seems to affect 16S amplicons sequenced with the Lib-A chemistry.

[Benh]

Hi all,

I'm wondering if other labs are having problem 5) mentioned in the original post about high trimming rates and short reads.

Probably since early 2013 many of our amplicons/16s sequencing runs suffer from high short reads. Samples improve with multiple rounds of clean up the rid of the primers (ampure + gel purification) but the sequencing throughputs are still nowhere as good as they were.

We have been using Lib-L LV for similar project last year and they were fine. As far as I know Roche only mentioned change in Lib-A emPCR reagent but not Lib-L.

Just wondering if other labs are seeing this as well and how are they coping with the situation.

[meimun]

Low enrichment = Uncoupled Beads?

Hi Everyone,

Until now, I have been free from bead enrichment issues, but recently my last 2 emPCRs had 1-2% enrichment. Remembering that this post mentioned that 1-2% is the "background" enrichment rate, and in combination with what I saw, I think I have a theory on what is happening, and I wanted to see if any of you noticed this as well.

Yesterday after bead collection, I was left with, again, only 1-2% enrichment, not the 3-fold higher that I was expecting. What I had noticed, though, was that there were a significant number of beads that were not "uncoupling" from the magnetic beads. Even though my Melt Solution was freshly made, I thought perhaps something was wrong with our 10N NaOH solution. So I remade the Melt Solution using a different stock, and tried again, splitting the beads in two and performing two melts. Each time I did the melt, I only got a tiny little pellet.

I was wondering how many beads I had lost, and how I could find out. It occurred to me that since magnetic beads bound to capture beads are heavier than magnetic beads alone, so I quick spun the tubes to separate the them. And surprisingly it worked. The magnetic beads stayed in suspension, and there was a brown and white pellet. I collected the supernatant and put it on the MPC. It was all solid brown with magnetic beads. I also resuspended the pellet in Enhancing Buffer and put it on the MPC. It was white and brown. I uploaded a pdf showing photos from the experiment of the uncoupled beads that I sent to Roche. Roche is now investigating the kits.

Has anyone else seen this?

Did you ever try sequencing those beads that enriched at 1-2%? If so, how did that go? I would find that I would sometimes get the same thing, but never any lower. As a test I did one reaction with no DNA and got about 1-2% enrichment. I think that's just the number of beads that stick no matter what, so any time I get enrichment values in that range I just assume that there's nothing there at all.

[vlee2]

Hi, here you go

We also tried to evaluate DNA capture beads after emPCR using almost identical procedure Moorpark has described, but the only drawback of this is that MinElute columns don't retain fragment of dsDNA 100 and below. For ssDNA this number is probably even lower. You may want to try NucleoTrap kit.

[Moorepark]

Hi, here you go

[meimun]

I also got a useful piece of troubleshooting- there is a step you can do where instead of discarding the first melt post bead recovery (pre-enrichment), you keep it and neutralise, and purify the ssDNA. This can be run on an RNA bioanalyser chip to see if it contains short fragments. I am happy to send on the protocol to anyone interested. It isn't really worth doing routinely I;d say because of cost, but it is for samples that have been problematic or if you're having problems with everything- as we were lately after a good 6 months of everything working smoothly!
Hope all this helps!

Hi Moorepark,

I think that is a genius piece of troubleshooting, and would love to try it myself. Can you upload it? Thanks in advance.

[Moorepark]

Hi all,
I too have been having similar problems, very low enrichment one day, very high the next. My LV reactions are nearly always ok, but have been having major problems with SV and MV. It got to the point where I no longer bothered doing a titration, just took my chances with LV- as I said, it usually works out, but the MV have been awful. THe last few MV I have done have all given me either really high enrichment, or obviously broken emulsions. After several telecons with sequencing service in the UK and Germany, and between them and 454 in the US, I have received some useful input. Firstly, as you know, some bead recovery reagents were faulty about 6 months ago, and a general recall was not made, it was only on a case by case basis. Apparently, I was still using some of the red-flagged beads- 93874520. These beads result in what looks like over-enrichment, a "sludgey" bead lettet during enrichment where it's difficult to get off the white beads without taking off brown.
The other useful (hopefully) piece of information I have received is that the arms of the tissuelyser need to be balanced- apparently if you only use one arm, or put emulsions of different sizes on (i.e. large on one, mv on the other) then it can cause your tissuelyser to destabilise the oil, leading to obviously broken emulsions sometimes but also to emulsions that can look ok but are not. We used to use one arm of the tissuelyser quite regularly with the old oil, without problems, but someone else on this forum has mentioned that the new oil is less stable so maybe there is some truth in this. Apparently stability vaires with container size, such that LV>MV>SV. I am going to try my MV again next week, putting half on one arm and half on the other, so I'll let you know if this improves things.
Lastly, I also got a useful piece of troubleshooting- there is a step you can do where instead of discarding the first melt post bead recovery (pre-enrichment), you keep it and neutralise, and purify the ssDNA. This can be run on an RNA bioanalyser chip to see if it contains short fragments. I am happy to send on the protocol to anyone interested. It isn't really worth doing routinely I;d say because of cost, but it is for samples that have been problematic or if you're having problems with everything- as we were lately after a good 6 months of everything working smoothly!
Hope all this helps!

[ATCGquest]

I am also experiencing the same problems with 454. The percent enrichment varies each time I set up the emulsion. Even if I set up the emulsion with the same DNA sample and at the same cpb a second time, the percent enrichment changes.

I was wondering if anyone else is using 16S V1-V3 amplicons for 454 and experiencing these problems.

[UofG]

I just put a sequencing run on with beads combined from two runs that both resulted in low enrichment. I will know tomorrow.

[ajthomas]

I am relieved to see many people having the same problems as I have been experiencing the past two months. Well I am not happy that your work is being compromised, but I have been banging my head against the wall trying to figure out the problem. I have done 10 different emPCRs in the past month, and 9 have resulted in 1-2% bead enrichment, despite the concentration of my libraries being acceptable. The only one that worked was an amplicon library (all the failed runs being shotgun libraries with the Rapid Library kit), and it worked beautifully.

Roche suspected the Rapid Library kit I have been using, and never once mentioned the emPCR kit, which is what I have been suspecting. They replaced the Rapid Library kit, and my lastest bead enrichment had the same result with 1-2% enrichment.

Is anybody willing to share their kit lot#s?

Did you ever try sequencing those beads that enriched at 1-2%? If so, how did that go? I would find that I would sometimes get the same thing, but never any lower. As a test I did one reaction with no DNA and got about 1-2% enrichment. I think that's just the number of beads that stick no matter what, so any time I get enrichment values in that range I just assume that there's nothing there at all.

[ajthomas]

Exactly the same I saw for GS Jr kits, which utilize IKA Turrax mixer. The recommended speed produced many vesicles with multiple beads, so I had to increase speed and mixing time twice as much to get those broken up. Eventually I found that the original recipe published (with silicon oil) works more consistently than the later recipe employing mineral oil.

In my case, I found that increasing the time beyond 5 minutes didn't make much difference, but increasing speed did, but only up to a point. At about 25 Hz the beads just formed a large clump that couldn't break up at all. The emulsion was ruined.

[UofG]

I am relieved to see many people having the same problems as I have been experiencing the past two months. Well I am not happy that your work is being compromised, but I have been banging my head against the wall trying to figure out the problem. I have done 10 different emPCRs in the past month, and 9 have resulted in 1-2% bead enrichment, despite the concentration of my libraries being acceptable. The only one that worked was an amplicon library (all the failed runs being shotgun libraries with the Rapid Library kit), and it worked beautifully.

Roche suspected the Rapid Library kit I have been using, and never once mentioned the emPCR kit, which is what I have been suspecting. They replaced the Rapid Library kit, and my lastest bead enrichment had the same result with 1-2% enrichment.

Is anybody willing to share their kit lot#s?