Using Nextera Indexes with Nextera XT Kit

[Angus's Microbiota]

Hello All,

I am planning to use a Nextera XT library prep kit (FC-131-1024) for my current project. The lab I work in already uses the other Nextera Kit (FC-121-1030) and therefore has the indices for that (non-XT) kit (FC-121-1011).

Does anyone know if the non-XT indices work for the XT kit? Illumina Technical Support said it isn't recommended, but they have heard of individuals "accidentally" using indices for the other kit and it still working. They couldn't recommended this option, but it "might" work.

I asked if the two kits (FC-121-1011 and FC-131-1001) were different and, of course, that information is proprietary.

Any help would be appreciated,

Angus

[nucacidhunter]

I have used both of them interchangeably with customised Nextera using third party polymerase for PCR and results were similar. Sequence wise they are the same. I think the difference is that S5XX primers (for XT) have been resuspended in different buffer from N5XX (Nextera). This could be to compensate for different composition of tagged DNA resulting from two kits where Nextera tagged DNA is resuspended in RBS but XT tagmented DNA is not purified and contains salts from tagmentation and naturalisation buffer.

[exo]

I have used both of them interchangeably with customised Nextera using third party polymerase for PCR and results were similar. Sequence wise they are the same. I think the difference is that S5XX primers (for XT) have been resuspended in different buffer from N5XX (Nextera). This could be to compensate for different composition of tagged DNA resulting from two kits where Nextera tagged DNA is resuspended in RBS but XT tagmented DNA is not purified and contains salts from tagmentation and naturalisation buffer.

Have you seen better results when you changed the polymerase?

[nucacidhunter]

I have worked with species that have 50% GC and performance was equal to Nextera. That polymerase is claimed to be more robust in amplifying low or high GC regions but I have not used it for amplifying tagmented DNA from organisms with extreme GC content.

[JVGen]

I have used both of them interchangeably with customised Nextera using third party polymerase for PCR and results were similar. Sequence wise they are the same. I think the difference is that S5XX primers (for XT) have been resuspended in different buffer from N5XX (Nextera). This could be to compensate for different composition of tagged DNA resulting from two kits where Nextera tagged DNA is resuspended in RBS but XT tagmented DNA is not purified and contains salts from tagmentation and naturalisation buffer.

Nuc, can you provide anymore information on using the Nextera XT indexes with the Nextera protocol? We're about to purchase a MiniSeq with the intention of running the reduced-volume reactions for Nextera library prep that I found published in S. Kryazhimskiy et al. I'd like to add more indexes per flow cell - I could run 384 and still have a surplus of coverage (I'm sequencing PCR-amplified viral genomes of 10 kb). However, the standard Nextera kit only has 96-indexes available, so I'm trying to figure out if I can use the Nextera XT indexes easily....

Thanks!

[SNPsaurus]

We use Nextera and Nextera XT indexes on Nextera libraries. Like you, we want to multiplex more than 96 samples in a lane. We were told by Illumina to do this, and we see no issues.

[JVGen]

Thanks SNP. You use the indexes interchangeably? No alterations? Just treat the XT indexes as the standard Nextera indexes?

Thanks much!

[SNPsaurus]

That's right. We prep a bunch of samples and just move from one set to the other with everything else the same.