MiSeq Pooling\multiplexing 240 samples using Nextera v2 Index

[elximo]

Hi everyone,

I am following Illumina's 16S protocol for sequencing and I use Nextera XT Index v2 adapters kit. I do my runs on MiSeq using 500 cycle v2 kit. The maximum number of samples I have sequenced in a same run is 64.

Currently, I am doing budget planning for a new project that would include almost ~240 samples. I prefer to sequence them all in the same run. The Nextera XT v2 index kit comes in 4 sets (A,B,C and D). Is it possible just to buy set A and D and use them together for the maximum theoretical limit of 384 samples or do I have to buy all 4 sets for that purpose?

To elaborate a bit, on a 96 well plate outlay there are 8 rows (S (i5) adapters) and 12 columns (N (i7) adapters). Illumina sells the Nextera XT v2 adapters in a combinatorial permutation: Set A (S1,N1), Set B (S1,N2), Set C (S2,N1) and set D (S2,N2), where Sx is 8 adapters and Nx is 12 adapters as found on this pdf: https://support.illumina.com/content...0002694-01.pdf

The information about the sets is here https://www.illumina.com/products/by...ra-xt-dna.html

Your help is appreciated.

[mastal]

I think that if you buy only 2 of the kits, you will get 192 different indexes, not quite enough if you expect to run 240 samples together.

[nucacidhunter]

Set A and D would give all 384 possible index combinations.

[elximo]

Thanks for both of your responses.

Originally Posted by mastal

I think that if you buy only 2 of the kits, you will get 192 different indexes, not quite enough if you expect to run 240 samples together.

The thing is Kit A and Kit D have different sets of adapters, otherwise the adapters overlap between others.

Originally Posted by nucacidhunter

Set A and D would give all 384 possible index combinations.

So this what I am speculating. However, I am trying to reach out to anyone who has actually just used A and D to do multiplex as many. Would the adapters like ligate properly if I use i7 from Set A and i5 from Set D or vice versa? Do not forget these are proprietary adapters by Illumina; they have structural changes.

Would there be any disadvantage for using just set A and D vs. using all four sets?

[thermophile]

I know you're part way through this project but I'd recommend you not use the illumina kit at all. follow the protocol in Kozich 2013, they provide indexes for multiplexing up to ~1500 samples.

[pmiguel]

It should work fine.

Remember there is no "ligation" with Nextera. The tagmentation adds "tag" sequences to the ends of the double-stranded breaks that are used in the subsequent PCR reaction to "step-out" from the tags, adding both indexes and flow-cell oligos.

Just plain old PCR oligos work fine. We have used a dual index set of our own devising for some sets of Nextera reactions. They were just 40 normal oligos ordered from IDT to give use 384 possible combinations.

--
Phillip

[auzzie599]

elximo, I am also considering just buying sets A and D in order to multiplex 384 samples. Did you have success with this strategy? or did you uncover any issues trying this?

[auzzie599]

Another option I am mulling over is whether to just synthesize my own nextera adapters to allow for greater multiplexing. pmiguel, what concentration of your adapters do you use going into the nextera preps? It seems Illumina does not want to disclose their adapter concentrations.

[pmiguel]

Originally Posted by auzzie599

Another option I am mulling over is whether to just synthesize my own nextera adapters to allow for greater multiplexing. pmiguel, what concentration of your adapters do you use going into the nextera preps? It seems Illumina does not want to disclose their adapter concentrations.

Yeah, good question since we will likely need to know this if we need to run more than 16 samples dual unique. I'd just check with a nanodrop first, see if the acetate salt is too high to allow you to resolve the absorbance at 260nm. Then maybe check them on an RNA nano agilent chip? Or, of course fluorimetry with an appropriate fluor.

You don't need to get the concentration perfect.

You also should check out Simone Picelli's work here.

--
Phillip

[Vinn]

Originally Posted by pmiguel

It should work fine.

Remember there is no "ligation" with Nextera. The tagmentation adds "tag" sequences to the ends of the double-stranded breaks that are used in the subsequent PCR reaction to "step-out" from the tags, adding both indexes and flow-cell oligos.

Just plain old PCR oligos work fine. We have used a dual index set of our own devising for some sets of Nextera reactions. They were just 40 normal oligos ordered from IDT to give use 384 possible combinations.

--
Phillip

Hi Phillips,

Do you have experience with 2-step PCR for Nextera XT V2 indexes? With the price of the XT indexing kit, we are looking for an alternative.

Illumina says base in adapter for N701 (i7) = TCGCCTTA and S501 (i5) = TAGATCGC in the adapter sequences leaflet. I guess we can use i5 indexes as they are. But should we reverse complement the i7 index sequences before using them with the reverse primer?

Thanks in advance!

[pmiguel]

Originally Posted by Vinn

Hi Phillips,

Do you have experience with 2-step PCR for Nextera XT V2 indexes? With the price of the XT indexing kit, we are looking for an alternative.

Illumina says base in adapter for N701 (i7) = TCGCCTTA and S501 (i5) = TAGATCGC in the adapter sequences leaflet. I guess we can use i5 indexes as they are. But should we reverse complement the i7 index sequences before using them with the reverse primer?

Thanks in advance!

For Nextera XT, we have always used the Illumina primers.
Your logic (above) looks sound to me.

--
Phillip