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October 1, 2016 (Vol. 36, No. 17)
The next-generation sequencing (NGS) market has been and continues to be dominated by Illumina, inflicting many researchers to bitch that the lack of competition is causing the rate of NGS development to gradual, as a minimum when it comes to can charge for short-study sequencing.
seeing that the launch of Illumina’s flagship HiSeq X sequencing platform, the can charge for a research-grade complete genome has held regular at round $1,000.
The simplest competencies competitors on the horizon, BGI’s Revolocity (based on expertise from finished Genomics), has considering that been shelved. youngsters, there have been a number of pleasing trends that promise to open up using NGS in different areas.
One area in which enormous development is being made is that of long reads, that are revealing areas of complexity in the genome and transcriptome that defy short-read sequencing. in the analysis of genomes, the basic issue is coping with repeated sequences of DNA. Repeats longer than the underlying read length will also be very complicated and even unattainable to uniquely map to the reference genome.One enviornment in which giant development is being made in next-gen sequencing is that of lengthy reads, which can be revealing areas of complexity within the genome and transcriptome that defy short-study sequencing. [Cosinart/Getty Images]
an identical challenges turn up in transcriptome evaluation. “Most genes include distinct alternative exons that are located farther apart in the mRNA than the examine length,” says Brenton Gravely, the John and Donna Krenicki Professor of Genomics and customized Healthcare at UConn fitness. “it is, therefore, unattainable to use it to accurately be aware of which isoforms are present in a particular pattern.”
The company that’s been getting the most attention currently around long reads is Oxford Nanopore, which first made their nanopore-based sequencer, the MinION, obtainable ultimate spring. however Oxford Nanopore has stretched the definition of a formal product launch, the company has certainly made its technology purchasable to a broad audience. Over 1,000 research businesses are the use of the MinION in their own laboratories.
inside a yr following its preliminary unlock, the MinION accelerated its output from lower than 100 Mb to over one Gb, primarily through a series of advancements in nanopore design and enzyme chemistry. And while the MinION rarely methods what Illumina’s platform offers when it comes to output, first-class, and price per Gb, the Oxford Nanopore instrument has two leading sights: read size and portability.
users have reported particular person reads of over 100 Kb, with general study lengths within the many tens of hundreds of bases. DNA input first-class and library prep appear to be the simplest precise factors limiting the read size—in case you feed the nanopores long DNA molecules, you're going to get long reads.
since the MinION is a handheld sequencer (with enthusiasts arguing over the premier dimension comparator—stapler, USB stick, smart phone, chocolate bar, and so on.), it's even possible to deliver the sequencer out to the box the place the facts is being accrued, in preference to processing samples after they have got been introduced returned to the laboratory.
statistics high-quality lags some distance in the back of Illumina, but it surely is improving over time. The newest R9 pore in “quickly mode” is getting accuracy rates up to ninety five% below the top of the line situations.
Pacific Biosciences, the leader in long-examine sequencing, has been producing some excitement of its personal. The enterprise introduced a massive new platform, the Sequel, at ASHG closing 12 months. This platform, which is the outcome of a collaboration with Roche, guarantees a few advancements over the computer it is changing, the RS II.
The Sequel, in assessment with the RS II, generates sevenfold greater output, occupies two-thirds less house, and requires one-half the capital expenses. Pacific Biosciences has struggled a bit of to get the platform utterly launched, dealing each with chemistry issues and provide considerations, in particular for the consumable SMRT (single molecule, actual time) cells. each concerns seem to had been resolved, so we are anxiously waiting for the primary client-generated facts.
The different participant to make waves during this house is 10X Genomics, with its GemCode™ expertise, that could generate linked reads from underlying short-read data. youngsters this expertise doesn’t have the entire advantages of true long reads (as an example, it can't aid with repeats which are longer than the underlying brief reads), the linked reads are in a position to generate greatly superior de novo assembly and phased reads.
The platform seems to work well enough that Illumina signed a co-advertising agreement with 10X Genomics past this yr, with ease if quietly signaling the death of Illumina’s personal Moleculo synthetic long-read know-how.
regardless of the entire growth, there are nonetheless concerns with long-read expertise, exceptionally for transcriptome sequencing. “one of the crucial considerations is the current throughput of those systems, but the bigger problem is that reverse transcriptases are only no longer processive sufficient,” explains Dr. Gravely. “a very outstanding reverse transcriptase—and even superior—direct RNA sequencing, can be video game changers.” Transcriptome researchers should be satisfied to note that Oxford Nanopore, which recently posted a paper on using the MinION to at once sequence RNA, has plans to launch a commercial package sooner or later.
a further utility that has been gaining loads of traction is single-cellphone genomics. except these days, all NGS projects (with most effective infrequent exceptions) have been carried out on a combination of cells, anyplace from hundreds of cells as much as 1000000 cells for a single sequencing library. If all the cells are completely homogeneous, as is often the case for somatic mobile genomes, this isn’t in reality an issue. although, there are several purposes the place the cells don't seem to be homogeneous, and pooling them collectively masks crucial tips.
for instance, tumor biopsies are notoriously heterogeneous, comprising both somatic and melanoma cells. And even the cancer cells from the equal tumor can have significantly distinct genomes. Pooling distinctive cells collectively will produce a combined genomic profile, complicating the interpretation. variations existing in exactly a small variety of cells may well be completely obscured.
a strategy some are beginning to attempt is to take a couple of single-telephone measurements from distinct locations in the biopsy. If ample individual measurements are made, a complete view of the tumor genome will also be built up.
Transcriptomics is yet another software it is starting to improvement from the single-cell method. The transcriptome can be tremendously variable from mobilephone to cellphone, and pooling cells together can imprecise the underlying variability in gene-expression patterns.
“handiest a few years in the past, the transcriptomics evaluation of a number of hundred single cells would have taken massive materials and time,” notes Richard Shen, a genomics scientist and entrepreneur who lately left Illumina to discovered RS technology Ventures. “Now, with the development of easy-to-use NGS library coaching methods and decreases in sequencing charges, the evaluation of a few thousand single cells isn't only feasible however also desirable in lots of purposes.”
For the training of single-cellphone libraries, a number of structures are available, together with the C1 from Fluidigm, which may technique 800 cells at a time, and the Chromium™ from 10X Genomics, which may technique as much as an striking 48,000 cells at a time. Such processing capabilities, besides the fact that children, can also not be to each investigator’s goal, as Dr. Gravely notes: “The throughputs of probably the most new systems or home made devices are very interesting, however for a person who works on splicing, the holy grail is single-cellphone, long-read sequencing.”
melanoma Liquid Biopsies
Yet a different extraordinary NGS building is the introduction of liquid biopsies for cancer prognosis. the use of NGS is fitting a bit events within the diagnosis of melanoma tumor biopsies, with groundwork drugs’s FoundationOne check being a major example—a targeted panel of 315 cancer-linked genes mixed right into a single look at various. These panel-based mostly tests are replacing the extra ordinary, single-gene checks.
cancer diagnostics are additionally transferring from tumor biopsies to liquid biopsies. as a substitute of extracting DNA from a bit of the tumor, melanoma liquid biopsies involve extracting either circulating tumor cells (CTCs) or phone free DNA (cfDNA) from the affected person’s blood. whereas strong tumor biopsies received’t go away due to the anatomical and cellular constitution tips they deliver, there’s lots of exhilaration around liquid biopsies on account of the versatility they present.
“From a melanoma-screening perspective, liquid biopsies are desirable as a result of they characterize an easy and accurate (potentially) presymptomatic verify that can be carried out mechanically to seize melanoma early,” states Gabriel Otte, CEO and co-founding father of Freenome, a liquid biopsy business. “From a prognostic or patient-monitoring viewpoint, they're unique for an identical reasons, however notably for as much as 30% of instances of cancer that can't be invasively biopsied for a variety of factors.”
As should be would becould very well be anticipated in a hot new field, a couple of new agencies are being fashioned, and greater dependent companies are retooling to take talents of what is expected to be a big and becoming market. At AllSeq we are tracking over 35 groups during this area.
Guardant fitness claims that its cancer liquid biopsy diagnostic was the first to reach the market when it was launched in early 2014. This diagnostic, called Guardant360, looks at 70 cancer genes. Freenome is taking a different strategy by sequencing the complete cancer genome. “We rely on our deep learning,” says Otte, “to work out the selected subregions that are most vital for making an accurate melanoma verify.”
Illumina, whose sequencing technology is being used via most of these businesses, has determined it desires to get within the video game as well through spinning out its own liquid biopsy enterprise, Grail. besides the fact that children, perhaps with a view to stay away from the look of competing without delay with its personal shoppers, Illumina reports that Grail will be focusing on presymptomatic screening, a a whole lot more challenging project that not one of the different companies has yet to center of attention on.
looking to the long run
while Illumina looks to proceed dominating the market for top-throughput brief reads, we are protecting our eyes out for developments in different areas. Oxford Nanopore has announced loads of upcoming launches, including its high-throughput nanopore sequencer, the PromethION. The business claims that it is going to have the throughput to compete with Illumina’s HiSeq X.
The PromethION is at present within the palms of at the least a few early-access valued clientele, but there is not any company launch date yet. Oxford Nanopore is additionally speaking about two gadgets for automating pattern coping with, the VolTRAX and the Zumbador, which promise to additional simplify the entire manner of sequencing DNA samples.With steady advancements in ease of use and savings in cost of next-gen sequencing operations, the boundaries to scientific adoption now are inclined to center extra on legislation and repayment. [jxfzs4/Getty Images]
we are able to even be looking to see how right away Pacific Bioscience’s Sequel platform gets adopted. it will probably birth bringing the charge of long reads down sufficient to drag some business faraway from Illumina. finally, we’re preserving a watch on clinical functions of NGS. With regular advancements in ease of use and discount rates in can charge, the limitations to scientific adoption now are likely to middle greater on legislation and reimbursement.
enhancing pattern tracking in Longitudinal stories
With the introduction of liquid biopsy assays to monitor treatment response of oncology patients in research reports, suitable monitoring of samples has develop into increasingly essential. at the onset of such programs, not less than three samples are analyzed for his or her genetic profile; tumor tissue, usual tissue, and circulating, cellphone-free DNA.
When performing sequencing on a large goal similar to total exome sequencing, a genetic fingerprint can be decided to verify that samples are effectively matched during information evaluation. besides the fact that children, when sequencing with small focused panels, a genetic fingerprint can not be produced, in keeping with Drew McUsic, an software scientist at Swift Biosciences.
“previously, researchers have utilized single nucleotide polymorphism primarily based arrays paired with LIMS techniques to safely song samples,” referred to Dr. McUsic. “These approaches depend on relevant labeling of diverse samples and records info to retain actual matches of all substances.”
looking for an necessary, extra genuine components of sample identification, Swift Biosciences developed the Accel-Amplicon™ Sample_ID panel. A genetic fingerprint offered with the aid of the 104 exonic and gender particular amplicons is ideally utilized as a low percent spike-in to any Swift amplicon-primarily based panel, such because the Accel-Amplicon 56G Oncology Panel v2, defined Dr. McUsic, including that this effects in sample identification from low depth sequencing of germline targets while nonetheless enabling excessive depth of insurance for somatic mutation detection.
“Such a technique offers a good, single-tube assay for analyzing somatic mutations in oncology specimens whereas generating the genetic fingerprint inside the equal sequence file,” he persisted. “As extra projects are designed to tune response fees in oncology sufferers, it should be important to opt for appropriate pattern tracking tools in these longitudinal studies.”The Accel-Amplicon 56G Oncology Panel v2 comprises Sample_ID goals spiked-in at low percentage, allowing for somatic mutation detection using excessive depth of insurance and sample identification of germline targets using low insurance depth.
Shawn C. Baker, is Co-founder and CSO of AllSeq, Inc. He can also be reached at shawn@