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October 1, 2016 (Vol. 36, No. 17)

The subsequent-technology sequencing (NGS) market has been and is still dominated by means of Illumina, causing many researchers to whinge that the inability of competitors is causing the fee of NGS development to slow, as a minimum when it comes to can charge for brief-examine sequencing.

considering that the launch of Illumina’s flagship HiSeq X sequencing platform, the charge for a analysis-grade entire genome has held steady at around $1,000.

The most effective advantage competitors on the horizon, BGI’s Revolocity (in line with expertise from complete Genomics), has seeing that been shelved. however, there had been a couple of enjoyable developments that promise to open up using NGS in other areas.

One enviornment in which tremendous progress is being made is that of long reads, which are revealing areas of complexity within the genome and transcriptome that defy short-study sequencing. within the analysis of genomes, the simple issue is coping with repeated sequences of DNA. Repeats longer than the underlying examine length will also be very elaborate and even inconceivable to uniquely map to the reference genome.

One enviornment wherein colossal progress is being made in subsequent-gen sequencing is that of lengthy reads, which are revealing areas of complexity in the genome and transcriptome that defy short-examine sequencing. [Cosinart/Getty Images]

an identical challenges happen in transcriptome evaluation. “Most genes include distinct alternative exons that are located farther apart within the mRNA than the read size,” says Brenton Gravely, the John and Donna Krenicki Professor of Genomics and customized Healthcare at UConn fitness. “it's, for this reason, unattainable to use it to precisely be aware of which isoforms are latest in a specific sample.”

long-study expertise

The business that’s been getting the most consideration recently round long reads is Oxford Nanopore, which first made their nanopore-based sequencer, the MinION, obtainable last spring. however Oxford Nanopore has stretched the definition of a proper product launch, the enterprise has definitely made its expertise accessible to a large audience. Over 1,000 analysis organizations are using the MinION of their own laboratories.

within a year following its preliminary liberate, the MinION improved its output from lower than a hundred Mb to over one Gb, basically through a series of advancements in nanopore design and enzyme chemistry. And whereas the MinION hardly ever methods what Illumina’s platform presents in terms of output, exceptional, and cost per Gb, the Oxford Nanopore instrument has two main points of interest: examine size and portability.

clients have said particular person reads of over 100 Kb, with ordinary read lengths in the many tens of heaps of bases. DNA enter nice and library prep appear to be the most effective true factors limiting the read length—if you feed the nanopores long DNA molecules, you're going to get lengthy reads.

since the MinION is a handheld sequencer (with fans arguing over the most excellent measurement comparator—stapler, USB stick, wise mobilephone, chocolate bar, and many others.), it's even feasible to convey the sequencer out to the field the place the facts is being gathered, in preference to processing samples after they have got been brought back to the laboratory.

statistics high-quality lags a long way behind Illumina, nonetheless it is improving over time. The newest R9 pore in “quick mode” is getting accuracy prices as much as 95% beneath the most suitable situations.

Pacific Biosciences, the chief in lengthy-examine sequencing, has been producing some excitement of its own. The company announced a huge new platform, the Sequel, at ASHG final yr. This platform, which is the influence of a collaboration with Roche, guarantees a few improvements over the desktop it's changing, the RS II.

The Sequel, in assessment with the RS II, generates sevenfold greater output, occupies two-thirds much less area, and requires one-half the capital charges. Pacific Biosciences has struggled slightly to get the platform entirely launched, dealing each with chemistry issues and supply concerns, specifically for the consumable SMRT (single molecule, actual time) cells. each considerations appear to had been resolved, so we are anxiously looking ahead to the primary consumer-generated facts.

The different player to make waves during this house is 10X Genomics, with its GemCode™ know-how, which could generate linked reads from underlying brief-study facts. although this technology doesn’t have all the merits of proper long reads (for example, it cannot support with repeats that are longer than the underlying short reads), the linked reads are in a position to generate radically enhanced de novo meeting and phased reads.

The platform looks to work well adequate that Illumina signed a co-marketing settlement with 10X Genomics earlier this year, readily if quietly signaling the loss of life of Illumina’s own Moleculo synthetic long-examine technology.

despite all the growth, there are nevertheless concerns with long-study technology, above all for transcriptome sequencing. “one of the most considerations is the existing throughput of these structures, but the bigger subject is that reverse transcriptases are only now not processive enough,” explains Dr. Gravely. “a very top notch reverse transcriptase—and even more desirable—direct RNA sequencing, could be game changers.” Transcriptome researchers could be chuffed to notice that Oxford Nanopore, which currently published a paper on the use of the MinION to directly sequence RNA, has plans to launch a commercial equipment sooner or later.

Single-phone Genomics

another software that has been gaining loads of traction is single-phone genomics. except these days, all NGS projects (with simplest infrequent exceptions) have been performed on a mixture of cells, anywhere from hundreds of cells as much as 1000000 cells for a single sequencing library. If all of the cells are fully homogeneous, as is generally the case for somatic mobile genomes, this isn’t actually an issue. despite the fact, there are a couple of applications the place the cells don't seem to be homogeneous, and pooling them collectively masks important assistance.

for instance, tumor biopsies are notoriously heterogeneous, comprising both somatic and cancer cells. And even the melanoma cells from the same tumor can have extensively distinctive genomes. Pooling distinct cells collectively will produce a blended genomic profile, complicating the interpretation. adaptations present in precisely a small variety of cells may well be absolutely obscured.

a technique some are beginning to effort is to take a number of single-phone measurements from different areas in the biopsy. If satisfactory particular person measurements are made, a complete view of the tumor genome can be built up.

Transcriptomics is one other software it is beginning to advantage from the only-phone approach. The transcriptome will also be highly variable from mobilephone to mobilephone, and pooling cells together can imprecise the underlying variability in gene-expression patterns.

“simplest a few years in the past, the transcriptomics evaluation of a couple of hundred single cells would have taken large elements and time,” notes Richard Shen, a genomics scientist and entrepreneur who recently left Illumina to discovered RS technology Ventures. “Now, with the building of effortless-to-use NGS library coaching methods and reduces in sequencing expenses, the analysis of a number of thousand single cells isn't handiest possible however additionally beautiful in lots of purposes.”

For the instruction of single-mobilephone libraries, several platforms are available, together with the C1 from Fluidigm, that may technique 800 cells at a time, and the Chromium™ from 10X Genomics, which could process as much as an magnificent forty eight,000 cells at a time. Such processing capabilities, despite the fact, may additionally not be to every investigator’s goal, as Dr. Gravely notes: “The throughputs of one of the crucial new platforms or do-it-yourself devices are very enjoyable, however for a person who works on splicing, the holy grail is single-telephone, long-study sequencing.”

melanoma Liquid Biopsies

Yet an extra terrific NGS development is the introduction of liquid biopsies for cancer analysis. the use of NGS is fitting just a little activities in the prognosis of cancer tumor biopsies, with groundwork drugs’s FoundationOne test being a main illustration—a targeted panel of 315 cancer-connected genes mixed right into a single look at various. These panel-primarily based assessments are replacing the extra common, single-gene checks.

melanoma diagnostics are additionally moving from tumor biopsies to liquid biopsies. as an alternative of extracting DNA from a piece of the tumor, melanoma liquid biopsies involve extracting either circulating tumor cells (CTCs) or cellphone free DNA (cfDNA) from the patient’s blood. whereas solid tumor biopsies received’t go away due to the anatomical and cellular constitution information they give, there’s loads of excitement around liquid biopsies because of the flexibility they offer.

“From a melanoma-screening viewpoint, liquid biopsies are pleasing as a result of they symbolize an easy and correct (doubtlessly) presymptomatic check that could be performed mechanically to seize cancer early,” states Gabriel Otte, CEO and co-founder of Freenome, a liquid biopsy business. “From a prognostic or patient-monitoring standpoint, they are exciting for identical reasons, but particularly for up to 30% of circumstances of melanoma that can't be invasively biopsied for lots of factors.”

As should be would becould very well be expected in a scorching new box, a couple of new groups are being shaped, and greater centered agencies are retooling to take expertise of what is expected to be a huge and growing to be market. At AllSeq we are monitoring over 35 businesses during this space.

Guardant fitness claims that its melanoma liquid biopsy diagnostic was the primary to reach the market when it become launched in early 2014. This diagnostic, called Guardant360, appears at 70 cancer genes. Freenome is taking a different strategy by using sequencing the whole cancer genome. “We count on our deep learning,” says Otte, “to work out the particular subregions that are most significant for making an correct cancer examine.”

Illumina, whose sequencing expertise is getting used with the aid of almost all these companies, has decided it wants to get within the video game as smartly by way of spinning out its own liquid biopsy company, Grail. youngsters, most likely in order to evade the look of competing at once with its own purchasers, Illumina reviews that Grail should be specializing in presymptomatic screening, a an awful lot extra challenging assignment that none of the other groups has yet to center of attention on.

trying to the long run

while Illumina looks to continue dominating the market for top-throughput short reads, we're holding our eyes out for developments in different areas. Oxford Nanopore has announced loads of upcoming launches, together with its high-throughput nanopore sequencer, the PromethION. The enterprise claims that it will have the throughput to compete with Illumina’s HiSeq X.

The PromethION is presently in the arms of as a minimum a few early-entry valued clientele, but there is no enterprise launch date yet. Oxford Nanopore is also speaking about two instruments for automating pattern coping with, the VolTRAX and the Zumbador, which promise to extra simplify the whole procedure of sequencing DNA samples.

With regular advancements in ease of use and reductions in cost of subsequent-gen sequencing operations, the boundaries to clinical adoption now are inclined to core extra on law and compensation. [jxfzs4/Getty Images]

we are able to also be trying to see how immediately Pacific Bioscience’s Sequel platform receives adopted. it may beginning bringing the charge of long reads down sufficient to drag some business faraway from Illumina. eventually, we’re holding an eye fixed on scientific functions of NGS. With regular advancements in ease of use and savings in charge, the obstacles to clinical adoption now are likely to center extra on regulation and compensation.

improving sample tracking in Longitudinal stories

With the introduction of liquid biopsy assays to video display medication response of oncology sufferers in research reviews, suitable monitoring of samples has become increasingly vital. at the onset of such programs, a minimum of three samples are analyzed for their genetic profile; tumor tissue, commonplace tissue, and circulating, phone-free DNA.

When performing sequencing on a huge target equivalent to entire exome sequencing, a genetic fingerprint can also be decided to verify that samples are correctly matched right through data evaluation. besides the fact that children, when sequencing with small centered panels, a genetic fingerprint cannot be produced, in response to Drew McUsic, an utility scientist at Swift Biosciences.

“before, researchers have utilized single nucleotide polymorphism primarily based arrays paired with LIMS systems to thoroughly music samples,” referred to Dr. McUsic. “These strategies depend on proper labeling of dissimilar samples and statistics files to keep accurate fits of all substances.”

attempting to find an critical, more exact components of pattern identification, Swift Biosciences developed the Accel-Amplicon™ Sample_ID panel. A genetic fingerprint provided by way of the 104 exonic and gender particular amplicons is ideally utilized as a low percentage spike-in to any Swift amplicon-based panel, such as the Accel-Amplicon 56G Oncology Panel v2, defined Dr. McUsic, adding that this effects in sample identification from low depth sequencing of germline pursuits whereas nevertheless enabling excessive depth of insurance for somatic mutation detection.

“Such a technique offers an efficient, single-tube assay for analyzing somatic mutations in oncology specimens while producing the genetic fingerprint within the equal sequence file,” he endured. “As extra tasks are designed to music response quotes in oncology patients, it will be essential to choose correct sample tracking equipment in these longitudinal stories.”

The Accel-Amplicon 56G Oncology Panel v2 contains Sample_ID aims spiked-in at low percent, permitting for somatic mutation detection using excessive depth of coverage and pattern identification of germline pursuits the usage of low insurance depth.

Shawn C. Baker, is Co-founder and CSO of AllSeq, Inc. He can also be reached at shawn@

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