Nextera is an Illumina NGS library preparation system that works very differently from the standard fragment library preps being used in most labs. Instead of the usual Illumina fragmentation, end-repair and adapter ligation, the Nextera enzyme mix is simply added to DNA for five minutes and a library is ready for PCR amplification. Sounds too good to be true? Read on and find out a bit more about how it works and why you might well be seeing it in many more papers.
Cutting and pasting
The Nextera technology was developed by Epicentre and uses a modified transposition reaction called ‘tagmentation’ (figure 1). Transoposomes have free DNA ends and insert randomly into DNA in a ‘cut and paste’ reaction. Because the DNA ends are free, this effectively fragments the DNA while adding on the sequences required for PCR amplification and sequencing. Two transposomes are mixed in equimolar ratios and each carries one of the two sequences required for library PCR.
An improved performance
Tagmentation is random, and both insertion events are required to produce an amplifiable molecule. The reaction is treated to remove bound transopsases and then PCR amplified ready for sequencing. Illumina purchased Epicentre in 2010 and have optimised the Nextera kits to improve performance across GC rich regions and provide a size-distribution more like a bead size-selected TruSeq library.
Figure 1: Illumina Nextera workflow showing how two tagmentations are required to produce PCR amplifiable library molecules ready for sequencing. (a) Transposomes integrate into genomic DNA. (b) Tagmentation produces amplifiable and non-amplifiable library molecules until transposomes run out. (c) The library is cleaned to remove Tn5 protiens bound to the ends of DNA fragments, then PCR-amplified to add flowcell compatible adapters and dual-indexes for multiplexed sequencing.
Let’s be accurate here
Because the Nextera protocol involves an enzymatic tagmentation, the method is very sensitive to input DNA concentration. Therefore accurate quantification is vital. The protocol from Illumina suggests using Invitrogen’s ‘QuBit’ fluorometric assay (link) which is specific for double-stranded DNA. Methods like Nanodrop or UV-spec can give inaccurate results as they also measure ssDNA, RNA and other contaminants. The size of the final library is determined by the ratio of DNA to transposomes. Each transposome can only tagment once, the Tn5 protein is left stuck to the end of the fragment until it is cleaned off before PCR. This means the reaction stops after a defined number of insertions.
Discussion of dilution
The kits from Illumina are tailored to a 50 ng DNA input to give libraries of around 400-500 bp including adapter sequences; by varying DNA input or transposome concentration the size distribution of the final tagmentation library can be tailored to your specifications. Some groups have started to discuss diluting the reactions in the same way as we used to do for Sanger sequencing.
Making Nextera XT libraries from 1 ng of DNA:The newest kit from Illumina is designed for small genomes (bacteria or viruses), PCR amplicons, and plasmids. The same fast workflow prepares libraries in just 90 minutes, and it is possible to go from DNA to data in a day using Nextera and MiSeq.
Exome capture with Nextera libraries: Recently Illumina combined the Nextera method with their exome capture kits to produce workflow that allows production of 96 exome libraries in just 3 days. The 50 ng DNA input generates enough library to perform multiple captures and still have library left over for whole-genome sequencing. The latest kits use Illumina’s dual-indexing strategy where PCR primers and used in combination to prepare libraries for sequencing (figure 2). Using this method it is possible to run 96 libraries in a single lane of sequencing making the $50 genome possible for smaller genomes.
Figure 2: Illumina’s dual indexing uses combinations of primers shown as white and orange tubes, the supplied stand helps with PCR set-up.
Does it work?
The method has been adopted by many labs and several publications have been peer-reviewed. The Shedure lab at University of Washington published one of the earliest papers demonstrating the performance of the Epicentre kits for Human genome sequencing, 96-plex bacterial genome sequencing and exome sequencing. There are very few papers comparing the technology to the standard methods though. There are at least two papers coming out very soon that used Nextera combined with Agilent capture and I’d expect many more to come now Illumina are selling kits combining Nextera with TruSeq capture.