My lab has been a long-time user of Illumina’s transposase exomes for the very simple reason that the 50ng input has been the lowest on the market for number of years*. This made it attractive for cancer samples where we are really limited on DNA availability; not simply because there isn’t 1ug for analysis, but more because once the DNA from a patient is gone it’s gone! So we’re using only a little and saving the rest for technologies of the future. However the Nextera prep’s big pain point has been controlling insert size, where over tagmentation leads to short inserts and too many bases lost to overlapping reads, adapter read-through (and PCR duplicates – but that’s not Nextera’s fault).
Last week Illumina released the latest generation of Nextera: Netxtera Flex (sounds a lot like NextFlex from Bioo). This uses a bead-bound transposase, this limits the amount of transposase in the tube and means the long un-bound DNA molecules can be removed from the prep after BLT transposition (Bead Linked Transposome not Bacon Lettuce and Tomato sandwich). Transposition takes just 15 minutes with the rest of the protocol, including PCR and washes, taking a total of just over an hour.
Because of the use of bead-linked transposase it is not necessary to quantify DNA before prep if starting with over 100ng – presumably this is because Illumina can QC the batches of BLT to make sure the amount of transposase present is more reliable in each prep, and the excess DNA is removed meaning the long molecules are not left behind. Unfortunately this also means that the kit now stacks up less well in comparison to today’s competition. This means library prep is incredibly simple and fast for people with DNA inputs between 100–500 ng, they can now save the time on accurate quantification of the input DNA sample, and normalization of the final yield is expected.
Where’s Netxtera Flexome, FleX-T, or Flex-MP
Unfortunately Nextera Flex is not yet available for use with the exome kits – there is only the Nextera DNA Flex Library Prep Kit for genomes (big and small). I’m hoping it’s just a matter of time before this happens. If Nextera Flex works as well as Illumina say then I can’t wait to try it out on exomes (I made up Flexome, I doubt Illumina’s marketing team will go for that cheesy a product name!)
There’s no Nextera Flex XT listed on Illumina’s site so small genomes and amplicons can’t use the ultra-cheap XT prep with the improved Flex workflow. Ultimately it’s going to have to stack up against methods incorporating UMI’s and it might be bait-sets that
Nor has a Nextera Flex Mate Pair kit been released. I’ve not thought too deeply about this but there was a time when mate-pairs seemed like they’d have a big impact on structural variant analysis and this has not been the case partly because the preps were such a pain. A Flex-MP with very low amounts of transposase on teh beads might allow for long DNA molecules to be tagmented. I’ve no idea how big a bead actually is but I suspect you could wrap multiple 10-20kb molecules around them and get big bits of DNA off at the end?
I know some of the people who’ve been working on Nextera over the years and this looks like a nice update for the method. Please release the exomes soon, and if you can squeeze UMIs onto them that’d be great!
*the most recent releases from the likes of Agilent, NEB, Kapa, etc have meant the competition is catching up so this might not be the best choice today!
NEBNext Ultra DNA uses 5ng-1ug DNA inputs.
KAPA HyperPlus uses 1ng-1ug DNA inputs.
- Agilent SureSelectXT HS uses 10ng-1ug DNA inputs…and includes UMIs!