Multiplexing >96 Samples with NexteraXT

[docbio]

Ok, so the current NexteraXT dual-indexing adapters allow for 96 combinations using a 8x12 setup. We routinely run 96 samples per lane, and honestly, we'd like to add more.

The dual-index system is obviously scalable, and it wouldn't seem to require much work on Illumina's part to expand it to 384 unique combinations (16x24). My sales rep tells me that the system was designed to be scalable but she hasn't heard of anything in the pipeline. My core tells me we're the only user multiplexing so many samples per lane, so I'm not sure the demand is there to push them to go to the trouble and expense of validating and producing more indices.

The few commercially available systems I've seen for 384 index combos all seem to be 5' index strategies or otherwise wouldn't be NexteraXT compatible. I won't contemplate switching back from NexteraXT to a sonication/end-repair/ligation/amplification method as long as I'm the person making the libraries. Has anyone found a method or a way to NexteraXT-compliant indexing beyond the 96 combinations available at present?

best,
docbio

[kcchan]

The Nextera indexing PCR primer sequences are published on their website. You can use their sequences as a basis for designing additional barcodes. Unlike the TruSeq adapters, it's just a standard oligo primer, so it's fairly cheap to make.

[james hadfield]

I'd seconddocbio's request for 384 barcodes ad not just for Nextera XT. ChIP-seq and RNA-seq can often require more than 96 samples and we try to make a superpool and spread it across the number of lanes required to generate a specific depth rather than just run a specific number of samples per lane.

[docbio]

The Nextera indexing PCR primer sequences are published on their website. You can use their sequences as a basis for designing additional barcodes. Unlike the TruSeq adapters, it's just a standard oligo primer, so it's fairly cheap to make.

Ahhh, but that's the rub. It's not just a "standard oligo primer" - the adapters have a forked structure and there is some proprietary modification to the adapters that Illumina's not talking about. I remember reading some threads on here speculating about the nature of the modification, I can't find them now, but I just called Illumina tech support and they verified there is an adapter modification that is proprietary.

So my fear is that I'll wind up with the standard Illumina adapters and my home synthesized adapters won't work as well, and I'll have constant balance issues - or worse, that somehow the home grown adapters will throw a wrench into the mix in general and interfere with the amplification step.

I'm not sure how many people are looking to push beyond 96 indices, and as NexteraXT was originally targeted for the MiSeq market, I get the sense Illumina doesn't see much demand for >96 indices. However, on the HiSeq, it is possible to multiplex even more samples at longer read lengths and decrease your per library cost considerably. Tell your sales rep and tech services that you want more than 96, if enough of us ask for it they might decide to release them commercially. I'd be amazed if they didn't already have additional validated index sequences in house.

TGIF
docbio

[james hadfield]

Hmm, I'm not sure about the amplification primers having anything special on them. We use standard desalted oligos from Sigm for our amplification of Fluidigm libraries and we have run up to 1536 samples in a multiplex.

[kcchan]

The Nextera adapters are not the same as the TruSeq forked adapters. The TruSeq adapters do have a bunch of modifications which make them harder to homebrew, but the Nextera adapters are just regular old PCR primers.

[Zebrafish]

The Nextera adapters are not the same as the TruSeq forked adapters. The TruSeq adapters do have a bunch of modifications which make them harder to homebrew, but the Nextera adapters are just regular old PCR primers.

So you can just make nextera adapters by IDT and sequence them directly or you still need to buy the kits from Illumina/nextera.

Thanks.

[docbio]

So I hear from my Illumina rep that 384 index combinations for NexteraXT should be coming in April 2014. We will see...

[bpgoll]

So I hear from my Illumina rep that 384 index combinations for NexteraXT should be coming in April 2014. We will see...

Hear that too..
We'll know a lot after it actually came..

[docbio]

Hear that too..
We'll know a lot after it actually came..

Have you heard an estimated price? Based upon the difference between 24 and 96, I'm expecting it to be a fairly linear increase...

[ustar]

The Nextera adapters are not the same as the TruSeq forked adapters. The TruSeq adapters do have a bunch of modifications which make them harder to homebrew, but the Nextera adapters are just regular old PCR primers.

Correct, we have tried IDT synthesized oligos and they worked! No need for Nextera XT index kit from illumina anymore.

[nucacidhunter]

Correct, we have tried IDT synthesized oligos and they worked! No need for Nextera XT index kit from illumina anymore.

Thats great news. Were oligos disalted or purified by HPLC or gel and what is final concentration in your PCR reactions?

[ustar]

Thats great news. Were oligos disalted or purified by HPLC or gel and what is final concentration in your PCR reactions?

We used HPLC purified oligo. Final concentration is 0.5uM in PCR reactions. I would suggest you test the primer concentration yourself though, coz we used it for single-cell application.

[Chief_Lazy_Bison]

I found this reply in another thread:

Hi,
I am using Nextera XT kit from Illumina but tried to order the adaptors from another vendor, since the sequence is disclosed. Unfortunately, I get sub-optimal results due to accumulation of primer dimers after enrichment PCR.
I then ran a Bioanalyzer to compare the adaptors from the XT index kit with my (unmodified) adaptors and found out that the Nextera´s migrate as a fragment ca. 10 bp slower than the others. Obviously, they must be modified (blocked) somehow to prevent the formation of concatamers/dimers......

It seems like there isn't a consensus on this yet, has anyone else had any experience trying to order their own Nextera XT index PCR primers?

[Simone78]

I found this reply in another thread:





It seems like there isn't a consensus on this yet, has anyone else had any experience trying to order their own Nextera XT index PCR primers?

And it is because of that discrepancy between home-made and Nextera adaptors that we now just buy them from Illumina. For single-cell RNA-seq there is no need to use 5 ul of each index primer! therefore we dilute them 1:5 before PCR, saving lots of money. I guess one could decrease them further, even down to 1:10. This, of course, works for single cell projects where the input for tagmentation is generally low (we perform tagmentation on about 500 pg DNA, often 100 pg or less). If you start from more, home-made adaptors should give decent results if they are not in huge excess compared to the template (or they will make concatamers if they are not blocked). Some titration might be needed.