*Important* Current state of the Roche 454 sequencing

[Aniki]

Hello,

I have not posted here for sometime now, but I thought I would go ahead and share some of my experiences with Roche 454 in the last half year or so. If anyone can chime in and/or relay their difficulties as well, that would be super.

1) 454 Pyrosequencing has been very difficult for me to do in the last half year or so. The titrations do not match up with the MV and LV emPCRs most of the time. There were always differences before, but it was manageable and minimal. The differences now are in the 10-20% range (both more and less). Therefore, it is very difficult for me to achieve desired enrichment values.

2) There have been some recalls with SV oil and potentially MV oil. This is despite the fact that I was assured certain lots were fine, up until the recall. If anyone is interested, I can probably look up the lots that were recalled.

3) Approximately 6 months ago, there were a lot of issues with the Bead Recovery Kit. The enrichment beads would enrich out null-beads as well as DNA amplified beads. This resulted in many false positive results with sequencing runs featuring 50% to 75% empty wells. I believe this issue has been fixed, but it has not been fixed to what was originally achievable prior to the issues.

4) Approximately 6 months ago I started to see certain emPCR kits resulting in 0% to 2% enrichment rates, despite "good" titration values. This could have been caused by faulty bead recovery kits, but I think it is important to bring this up as well.

5) The most pressing issue (I believe), is the fact that we are seeing a lot of trimming as well as short reads. This started when the new emPCR kits phased in. My reads are no where near as good as before. However, this is possibly just the new reality.

As you can see, I have been having some issues. Our Roche FAS has been very helpful but despite her efforts we are unable to resolve the issues. We are constantly assured that we were the only lab experiencing these issues, but I do not believe this to be true. It has come to a point where I am unable to do any Roche 454 runs until this has been resolved.

Titrations do not work, and if they do work they do not correlate with the LV reactions. In the last couple runs I have resulted to "guesstimating" the cpb and achieved modest results. However, I do not intend on repeating this.

I hope things are running better for you guys. If not, please chime in.

Thank you.

[yaximik]

Nope. I started seeing the same problems last spring, but with GS Jr kits, which indicates that the problem is quite deep. They had to replace about 10 emPCR and bead recovery kits, but it did not fix the problem. I think they have no idea where is the problem, but it may be well in emPCR kit, not downstream from it. Increased formation of primer dimers during emPCR can create a lot of mess, for example, and some enzymes do it more then other. I got so frustrated I quit using GS Jr with dozens of kits stocked still in the lab.

[Anthony.287]

1) Definitely agree. The ratios of aqueous : oil do not match up across the kits, and this seems to have an effect. I've compensated by making sure to use the same lot numbers and same size kits for both titrations and bulk sequencing. It's a pain, but better than having to start over.
2) After observing ~25% emulsion breakage last week, alerted our FAS and we are having all our MV oil, lot 93885320 replaced. We're not the only ones.
3) I've managed to avoid this one, somehow.
4) I've noticed this too. In some instances I needed 10x to 100x more DNA to get usable enrichment with libraries that had sequenced very well in the past.
5) I've not seen this. I wonder if it is a software issue; we're still running 2.5.3.

[ajthomas]

I've been fighting with the same issues. It has gotten a little better in the last few months, but the issues still remain and I can't get as good results as I did before. I too believe the issues to be somewhere in the emPCR procedure.

Regarding the MV and LV enrichment results not correlating with SV: I was consistently getting higher titration results with the MV kit than SV, and I had to redo many emPCRs as a result. I began inspecting the emulsions carefully after thermocycling and found that some wells looked somewhat broken. Having never had broken emulsions before, I wasn't sure, but there was a think clear layer at the bottom of some wells. I looked at the emulsions under a microscope and found that some was still intact and some broken. Near the bottom of the well I had a lot of vesicles with many (sometimes several dozen) beads. I looked at the emulsion right after shaking it, and found that the emulsions weren't breaking during thermocycling; they were never made properly in the first place. After getting new tube holders from Roche and trying several variations of shaking speed and time, I was never able to get emulsions that looked as good as what I got with the SV kit. The solution I found that works is to split the MV oil into 600 ul aliquots in 2 ml tubes and do three SV reactions instead of 1 MV reaction. I do the initial 2 minute shake in the MV tubes, then transfer to 2 ml tubes and follow the SV protocol from that point on.

I don't know what's going on, but it needs to be fixed soon. It was working fine before they made some changes. I don't understand why they can't just go back to what they were doing before those changes.

[Krisztab]

Both (completely separate) labs in Germany we have been sending samples for pyrosequencing are complaining about problems ever since they have received the new kits. Roche acted as if it was a local problem of those labs, but it is now quite obvious that this is not the truth. What I believe is that they have released a kit under development and now they are testing it for free on our expenses.

I guess I don't have to explain what does it mean to have "few months" of delay for a PhD student.

So I am also looking for further proof these are not isolated issues.

[NathalieA]

since I begin on 454 pyrosequencing, we have some problems between SV, MV and LV.
Now, we use only MV and LV kits, because titration in SV was not reproduce in MV or LV scale.

We check the lot number for emPCR reagent and Bead Recovery very carefully, because the amount of enrichment beads is different between kits (enven if the lot number is the same!). So we check the number of beads and kits are classify according to the quantity of beads.

Regarding emulsion and broken emulsion, I think I have never seen yet.
But We have usually a translucid ring at the top of emulsion. Our FAS told us that it is normal since the new oil.

It is a little bit stressed to cross finger at each emulsion regarding to the number of library we put!

[yaximik]

after shaking it, and found that the emulsions weren't breaking during thermocycling; they were never make properly in the first place.

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.

[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?

[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.

[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.

[UofG]

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

[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.

[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!

[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, here you go