Nextera Success?

[cwivagg]

Hello,

I'm trying to use an old Nextera kit from before the company was acquired by Illumina. I have tried using the kit's HMW buffer on 15 ng of the included lambda control DNA.

After tagmentation and limited-cycle PCR, I get the attached size distribution. This is not anything like the size distribution I expected from the rather terse instruction manual, nor is it similar to the ones pictured here:

http://epicentral.blogspot.com/2010/09/size-distribution-of-nextera-illumina.html

Would the correct interpretation be that tagmentation failed or worked only poorly, or am I looking at something else entirely here? Besides bioanalyzer traces, what are other standard controls I might do to validate my library? I've thought about trying to PCR up random fragments, but it seems difficult. I'll appreciate any input at all.

Carl Wivagg

[NextGenSeq]

The kit has got to be expired by now if it was made before Illumina acquired Epicentre a couple years ago.

[cwivagg]

Thank you for this observation... it may be expired. I am more interested, though, in interpreting this result to determine what component of the kit has failed. I was hoping someone who has a bit of expertise in library preparation might be willing to offer a guess, or suggest controls. I would rather learn to troubleshoot on an expired kit than waste expensive new reagents.

Carl

[Olaf Blue]

Hi, do you have a lot number for that kit? Expiration is certainly the main consideration and the Transposomes (Nextera Enzyme Mix) probably went bad.

[SeqR&D]

I agree...it has to be the enzyme...everything else is buffer.

[koadman]

If you really want to use this kit you could try using only 1-2ng input to see if the fragment size distribution gets in the 200-300 range.

[SeqR&D]

If you buy a new kit, and run conditions where you replace one of the new reagents with one of the old reagents...then you can see which one is the trouble maker. It sounds like you need a new kit anyways.

what do you mean, "standard controls to validate your library"?

[rnaeye]

I believe that Nextera has short shelf life due to short half-life of transposon complex. If the kit is not fresh, you need to optimize fragmentation conditions again, such as using more enzyme etc. I went back to regular library prep protocol. Transposase(s) does not seem to be very stable.

[cwivagg]

If you buy a new kit, and run conditions where you replace one of the new reagents with one of the old reagents...then you can see which one is the trouble maker. It sounds like you need a new kit anyways.

what do you mean, "standard controls to validate your library"?

Regarding controls, I am wondering if there is anything I can do beyond a Bioanalyzer run. Do people try to TOPO-clone fragments to verify proper end-tagging, or anything like that?

Thank you everyone for your thoughts. I have since bought another Nextera kit and observed much better fragmentation. I am thinking about going back to the original kit and trying a much longer incubation to see if I get any fragmentation, as one responder suggested, since the consensus seems to be that the transposome is probably decayed.