There may still be room in NGS for innovation in workflow
A few years ago Illumina’s Scientific Affairs team put together a gigantic wall-poster of all the different types of library preparation methods for next-generation sequencing (NGS). Today there are no less than three posters (one for DNA, one for RNA, and a third for single-cell), and you are free to peruse them here. These are enrichment methods to answer specific scientific questions – upstream of the library construction process itself.
A few words about the NGS library preparation process
The starting material DNA can come from a variety of sources. In its most simple case, white blood cells (WBCs) are purified from venous blood via centrifugation, and genomic DNA prepared via any one of a number of methods (from manual phenol-chloroform extraction to silica glass fiber such as QIAGEN’s to magnetic bead purification such as Roche’s MagNA Pure). In other cases one of the hundreds of enrichment protocols references in the aforementioned posters could be the starting DNA, in still other cases the starting DNA can be long-range PCR products, or metagenomic DNA mixtures.
From all these samples the DNA needs to be fragmented to an average 300 base-pairs in length, typically via ultrasonication (Covaris manufactures systems to do this) or through enzymatic methods. After fragmentation, the ends of the DNA molecules need repairing (also called polishing) and A-tailing via an enzymatic method. After the end-repair and tailing reaction, the samples need to be purified, to remove enzymes, unincorporated nucleotides and salt, typically with a magnetic-bead cleanup step and sample transfer.
The P7 and P5 Illumina sequencing adapters are then ligated onto the ends of the DNA molecules, and then another magnetic-bead cleanup step performed, again to remove the ligase, nucleotides and residual reaction buffer.
Additional master mix plus adapter primers in a PCR step adds on sample identifying single- or dual-indexing sequences to the outside of the P7 and P5 adapter sequences, and then cleaned up once again.
This entire process takes from 2.5 hours to 4 hours, and this is starting from your DNA sample (i.e. does not include other pre-library enrichment such as ChIP or cDNA synthesis).
RhinoSeq’s Revolutionary workflow
A startup company SeqOnce (based in Pasadena California) has invented a streamlined method of preparing these libraries that does away with separate reactions and purifications. In a single well, you start with a low input amount (5 ng and can go lower) and add a reagent to fragment for 8 minutes, then ligate the P5/P7 adapters for 12 minutes, then (optionally) you add the indexes via PCR for 20 minutes, and finally you implement a single magnetic-bead cleanup.
This is in contrast to the other methods that have intermediate cleanup steps – which is where some of the time-savings comes in – and avoiding additional sample transfers, which saves on consumables (i.e. plasticware such as tips, plates, strips or tubes).
Their kit is so simple it has only five reagent vials, and I am told that the GC bias is minimal, competitive with other library preparation kits in the market. This area has many companies providing kits for library preparation – Nextera DNA Flex from Epicentre/Illumina, HyperPlus from KAPA/Roche, NEBNext Ultra from New England Biolabs, and Swift Biosciences – but a combination of time- and labor-savings is significant, as well as an aggressive $20/sample pricing per their website.
Areas to improve
As SeqOnce is just getting started they are working on expanding their offering beyond WGS/WES library preparation, to both analysis of RNA for RNA-Seq (currently a much longer protocol that is difficult to automate) as well as FFPE analysis (where both low-input requirements and ease-of-use in a clinical setting are both paramount).
In addition, their website currently does not have data at this point showing their evenness of coverage as a function of GC content, but I have seen encouraging results, and am told that at the upcoming ABRF conference in Houston Texas March 23-26 they will be exhibiting there.
Worth checking out
If you are looking at speeding up your NGS laboratory operations, SeqOnce is worth a look. From their website they have an inexpensive 8-sample $160 kit, making it easy to try out, and a handy FAQ here. You can contact them via email here.