Suggestions to optimize IonTorrent workflow

[gedoardo83]

Hi everybody,

We are planning to acquire an Ion Torrent PGA and reading around the web it seems that the sample prep is the critical step for this technology.
With the aim to rapidly get good results we are evaluating which accessories will be useful to optimize and speed up the sample preparation.

Apart of the Bioruptor sonicator suggested by Applied itself and the OneTouch system, which other complementary equipments do you suggest to buy in order to make the sample prep as easy and fast as possible?

Thanks a lot!

[Hiro.Protagonist]

The bioruptor isn't the best for fragmenting DNA, it actually performs about as good as the fragmentase from NEB.

If you have access to a Covaris for shearing, I would definitely use that over the bioruptor.

An Agilent 2100 bioanalyzer is essential and a Qubit pairs well.

[TonyBrooks]

The bioruptor isn't the best for fragmenting DNA, it actually performs about as good as the fragmentase from NEB.

If you have access to a Covaris for shearing, I would definitely use that over the bioruptor.

An Agilent 2100 bioanalyzer is essential and a Qubit pairs well.

I would second the Covaris. I would also see if you could justify purchasing an E series model. We have the S series and it's very time consuming the moment you begin fragmenting more than a handful of samples - in only does one at a time. I would say that fragmentation is now becoming the bottleneck of our sample processing.

[Northeaster]

Anyone has tried the new enzymatic fragmentation method offered by Ion Torrent in its Xpress Fragment library kit? How does it work?

[Coyk]

Sorry for the delayed response. I have tried the IonShear protocol several times now with no success on my sample. The first two times I used 1 microgram starting material and I ran an E.coli standard as well. The E.coli sheared fine both times but my sample didn't at all. I then used 100ng starting material and tried increasing incubation times and still no success. Technical support seems to think that maybe my DNA has EDTA in it though I used a Qiagen DNeasy kit to extract it and the final elution is in EB buffer which is EDTA free. As of now, I haven't been able to make it past this first step.

[ECO]

What organism is your sample from? Something highly methylated?

[Coyk]

It is DNA from coral. I don't know if its DNA is highly methylated or not. I ran the exact same sample through the 454 Rapid Library protocol and it worked fine and subsequently got good results from a 454 Junior run.

[SeqAA]

Sorry for the delayed response. I have tried the IonShear protocol several times now with no success on my sample. The first two times I used 1 microgram starting material and I ran an E.coli standard as well. The E.coli sheared fine both times but my sample didn't at all. I then used 100ng starting material and tried increasing incubation times and still no success. Technical support seems to think that maybe my DNA has EDTA in it though I used a Qiagen DNeasy kit to extract it and the final elution is in EB buffer which is EDTA free. As of now, I haven't been able to make it past this first step.

EB can contain EDTA.

you have something inhibiting enzymatic activity. If the Ion Shear isn't working, chances are the rest of the library prep will not work either.

[lek2k]

You may also need a way of size selecting your fragments.
We are currently trialing the Invitrogen E-gel Size Select system. It's very fiddly but seems to do the job.

[Coyk]

EB can contain EDTA.

you have something inhibiting enzymatic activity. If the Ion Shear isn't working, chances are the rest of the library prep will not work either.

That's weird! The Qiagen website says that it's "10 mM Tris-Cl, pH 8.5". Also, I find it remarkable that I could put my sample through the 454 Rapid library prep with half the amount of starting material and have no problem. All this to say, we would love a Covaris to drop into our laps soon.

[lek2k]

EB can contain EDTA.

you have something inhibiting enzymatic activity. If the Ion Shear isn't working, chances are the rest of the library prep will not work either.

Why don't you just elute in (treated) water to be on the safe side. If IonShear still doesn't work, you should run a NEB fragmentase experiment in parallel. Fragmentase is dirt cheap so worth a try.

[Hamid]

Hi CoyK,

With mechanical shearing methods, you do not have to worry about the sample DNA source, and concentration. I am sure we can arrange to shear your 1-2 samples for you in TE or EB to show that it is not your sample. Please contact me if you like.

Thank you

Hamid

[ScottC]

Buffer EB, if you source it from Qiagen, should NOT contain any EDTA. We use it for almost all stages of the Illumina sample preps, which are mostly enzymatic reactions.

[Apollo704]

I second the E-gel size select, or a similar tech such as the pip'n prep.
A Guava is particularly useful too in that you will be able to determine % templated, and also the final bead quant. two things it's good to know before loading a chip.

[aleferna]

IonShear / One Touch performance??

We are trying to make a decision between buying a IonTorrent or a MiSeq, the problem is that the MiSeq has much better downstream stuff(Ie Nextera / no emPCR). I like the IonTorrent, but there is no information regarding the performance of the One Touch or the IonShear lib prep. Does anybody have any experience with these, do they work at all? I mean no sense in buying a Ion Torrent if you don't have a Covaris right?