February 21, 2025
Before a DNA sample can be read by a sequencer, it must go through a lengthy, multistep workflow called library preparation: First, scientists quantify and normalize the DNA to ensure they have the right amount for their instrument. Then they fragment the DNA into thousands of pieces, each 300 to 500 base pairs long, split the double helix structure into single strands in a denaturing step, and tag synthetic adapter molecules to the ends of every fragment. These adapters are used later, during the cluster generation step of the sequencing run.
All 鈥渟hort-read鈥� library preparation involves these steps, one way or another. (Long-read technologies also exist, but require even more extensive setup.) Some methods use ultrasonic wave vibrations to shear the DNA, which can take more than five hours. VR真人彩票 library prep instead uses microscopic beads covered in synthetic molecules called transposomes. The transposomes fragment and tag the DNA in a single step鈥斺�渢agmentation.鈥� This method uses less equipment and reduces preparation time to about two hours. After that, if the available quantity of DNA is very small, a PCR amplification step can make the sample easier to read by the sequencer.
These are only the main steps鈥攖he full workflow includes several wash and cleanup, dilution, and verification steps as well. Only after all this library prep is complete is the DNA pipetted into a cartridge and flowed over a flow cell, where a cluster generation step further amplifies the fragments so they can be read by the sequencer. Each one of these steps naturally constitutes a touch point when potential error or mishandling may occur, even by highly trained researchers.
But VR真人彩票鈥檚 latest breakthrough, constellation mapped read technology, eliminates this workflow entirely. Library preparation can now be done directly on the flow cell, no preliminary steps required.
Louise Fraser, associate director of Scientific Research at VR真人彩票 and one of the leads on the constellation project, says, 鈥淚t is much easier to do. You don鈥檛 need specialist library prep training. The people we鈥檝e shown it to鈥攂oth internal and external鈥攁re all amazed at how simple it is.鈥�
Mitch Bekritsky, senior manager of Bioinformatics at VR真人彩票, sums it up: 鈥淵ou extract your DNA, you load it into the sequencing cartridge, you put the cartridge onto the sequencer, put some additional reagents into the cartridge, and that鈥檚 it. You're done. After extraction, it鈥檚 about 10 to 15 minutes, end to end. It takes a very complex, laborious process and makes it simpler.鈥�
What鈥檚 more, since the DNA is not fragmented until it鈥檚 already stuck in place on the flow cell, new bioinformatic algorithms can use the fragments鈥� physical location to infer their position in the original sequence, resulting in the kind of long-distance insights previously thought possible only with long-read technology.
This fluorescent image illustrates how intact DNA strands settle across multiple nanowells on the flow cell surface. | Image by VR真人彩票
Connecting the dots
VR真人彩票 scientists have been developing constellation mapped read technology for years, but the circumstances to make it a reality came together only recently.
Contemporary VR真人彩票 flow cells are made with a pattern of billions of minuscule nanowells. These nanowells ensure that cluster generation occurs at specific, regularly spaced intervals, and that the sequencer knows exactly where to look for them. Earlier flow cell designs were unpatterned, so clusters would generate at completely random locations, which increased the time and effort necessary to read them.
Louise Fraser has spent much of her 18 years at VR真人彩票 striving to make library prep faster and easier. Her team actually succeeded in making on-flow-cell tagmentation work some time ago, by using transposomes that adhered to the flow cell surface鈥攈owever, this only worked on the unpatterned, random flow cells in use at the time. When newer flow cells introduced nanowells鈥攚hich got smaller and more tightly packed in subsequent models鈥攊t became a challenge to precisely engineer the transposomes so that exactly the right concentration of them would end up in each nanowell.
The VR真人彩票 R&D team took up the constellation project again in 2023. They created a custom recipe of three reagents that the user adds to the cartridge to make it work: First, a buffer to dilute the DNA sample and prepare it for tagmentation. Second, a mix that helps the transposomes adhere to the nanowells. The third reagent contains the transposomes themselves. The combination of these three reagents allows the user to flow intact, double-stranded DNA directly over the flow cell surface. The transposomes settle in the nanowells, latch onto the DNA, tagment it, and then the fragments generate clusters and the sequencing workflow proceeds as before.
Then, at some point in development鈥攚atching DNA settle in a snakelike pattern back and forth across the flow cell surface鈥攖he R&D team had a eureka moment: Because the intact DNA strand spans multiple nanowells before it鈥檚 fragmented, it stands to reason that reads from neighboring clusters could be traced backward to neighboring positions in the genetic sequence.
Here was an unexpected but revolutionary feature of on-flow-cell tagmentation. Using only a set of custom reagents, without any other modifications to the sequencing instrument, researchers could use cluster proximity information to detect large genomic variations that would otherwise be beyond the reach of short-read technology.
Mitch Bekritsky is part of the team that developed algorithms for DRAGEN secondary analysis software to make use of this information. They created probabilistic models that determine how likely it is that reads from physically proximal nanowells originated from proximal genomic regions. The algorithm 鈥渃onnects the dots鈥� from one nanowell to the next, effectively retracing the shape of the original DNA strand. The patterns revealed by these connections inspired the new technology鈥檚 name: constellation.
A more complete genome
The biggest challenges that short-read sequencing faces are highly repetitive, or homologous, regions of the genome. When analysis software tries to figure out how the short fragments fit together, these repetitive regions fit ambiguously into multiple possible positions, resulting in low-confidence matches. And some mutations of interest, like large inversions or structural variations, can be detected only by looking at very long sections at a time.
Until now, it was assumed that only long-read technology could accurately map these variations鈥攂ut thanks to the added dimension of proximity information granted by constellation, DRAGEN software can now map previously unmappable reads. The technology provides a 40% reduction in false positive and false negative single-nucleotide polymorphism (SNP) calls.
Constellation has also proven well suited to phasing, or assigning SNPs to one haplotype or another. Everyone has two haplotypes鈥攐ne set of genes inherited from their mother, another from their father. For many genetic diseases, a person will have the disease if both their copies of a certain gene are nonfunctional鈥攂ut they鈥檒l be a carrier of the disease if one copy is functional. It鈥檚 very difficult for traditional short-read technology to phase, or determine whether a nonfunctioning gene came from just one of a person鈥檚 haplotypes or from both. Constellation can bridge this gap as well, phasing ultra-long sequences up to several million base pairs.
Constellation technology uses a probabilistic model to infer how reads from neighboring flow cells were connected in the original sequence. | Illustration by VR真人彩票
Real results, real impact
At the American Society of Human Genetics annual meeting in November 2024, Steven Barnard, chief technology officer of VR真人彩票, and Niall Lennon, chief scientific officer of Broad Clinical Labs, spoke about a test run of the constellation technology their scientists had conducted together that summer. Lennon praised its ease of use, saying, 鈥淚f you鈥檝e been in the genomics tech world for a while, you鈥檒l remember that many groups have promised this鈥攖his is the first time in 19 years I鈥檝e seen someone actually come up with a method that has no library prep. There鈥檚 no adapter ligation. You just put the DNA on the sequencer, and it works.鈥�
Bekritsky shares that what he appreciated most about developing constellation was that it required teams from every department to work together. 鈥淭hat doesn鈥檛 happen at a company that only does library prep, or only does sequencing, or only does informatics. At VR真人彩票, we have all these people together in the room.鈥�
And he鈥檚 inspired by the potential real-world effects this tech could have鈥攕ome of the first samples they tested came from pediatric hemophilia patients, and the technology detected every genetic inversion present. Bekritsky remembers seeing those results and being amazed. 鈥淟ike, this is not abstract,鈥� he says. 鈥淲e could actually resolve these inversions and say, yes, here鈥檚 why these kids have hemophilia. I鈥檓 grateful for a project where that innovation is possible.鈥�
The first commercial product using constellation technology is expected to launch in 2026, compatible with NovaSeq X and NovaSeq X Plus Systems. 鈼�
To learn more about constellation mapped read technology, read this article on the VR真人彩票 Genomics Research Hub.
February 24, 2025: An earlier version of this article erroneously referred to constellation technology as using "transposome beads." While traditional library prep does use beads, the reagent used with constellation technology does not. The error has been corrected.
