AN EXPLOSION OF NEW SEQUENCING TECHNOLOGIES Insidegate250.com/tc2/BITW-eBook-Sequencing...

AN EXPLOSION OF NEW SEQUENCING TECHNOLOGIES

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The Quarterly eBook of Bio-IT World’s Most Trending Articles

InsideNote from Allison Proffitt

A New Beginning Semiconductor Sequencing

A Worthy Sequel: PacBio’s New Sequencing System

Thermo Fisher Clarifies Its Vision for Sequencing with Release of Ion S5 Instruments

BGI Retools Complete Genomics Technology for Its New High-Throughput Benchtop Sequencer

QIAGEN Releases GeneReader for Clinical Sequencing in Cancer

Direct Genomics’ New Clinical Sequencer Revives a Forgotten DNA Technology

Jolly Jay Flatley Ushers in Another Big Year for Illumina

NanoString Reveals Novel Sequencing Method for Cancer Assays

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In the mid-2000s, before Illumina solidified its technology as the default choice for DNA sequencing, a startling variety of scrappy little companies were working on their own peculiar ideas for quickly decoding the DNA molecule. The past year has seen a surprising revival of this pioneering spirit, as a huge number of new sequencers were introduced using technologies both new and old. Pacific Biosciences made its biggest play yet with the Sequel; life science giants BGI and QIAGEN entered the field; smaller companies like Direct Genomics and NanoString toyed with their own novel approaches; and even Illumina is striking out in a new direction with the Firefly project. Bio-IT World has covered it all.

Allison Proffitt, Editorial Director, Bio-IT World and Clinical Informatics News


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BY AARON KROL | FEBRUARY 2, 2015

In December 2010, Ion Torrent of Connecticut launched the first commercial product based on a new technology called semiconductor sequencing. The Ion PGM, sold at half the price of its nearest competitors, was taking a bold step into reading DNA electronically. Unlike other gene sequencers whose bulky optical systems looked at fluorescent dyes fixed to DNA molecules, the PGM used an array of microchips that could read electrical signals given off when DNA binds to a nucleotide. Ion Torrent, which

had been acquired by the larger Life Technologies just months before and had the resources to move aggressively into the genomics market, hoped to seize the momentum in a rapidly shifting field, and become

the sequencer of choice for genetics labs around the world.

Things have not quite panned out for semiconductor sequencing in the years since. Ion Torrent followed the Ion PGM with the more advanced Ion Proton: both have mostly fizzled under heavy competition. After a second acquisition by Thermo Fisher Scientific, the company is now racing to keep its customers, increase the speed and throughput of its instruments, and get the nod from the FDA to sell its technology for clinical use, but is lagging behind market leader Illumina on every count.

Chris Toumazou, CEO of DNA Electronics, holds a Genalysis chip. Image credit: DNA Electronics

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Chris Toumazou, who pioneered the field of semiconductor sequencing at his company DNA Electronics (DNAe), says he’s not surprised Ion Torrent has struggled. “Ion Torrent are competing with Illumina, who have got full genomes right up to a high spec,” he says. Ion Torrent uses the same basic platforms as Toumazou’s company — Thermo Fisher has a non-exclusive license to use DNAe’s patents for genome-wide applications — but the two companies have very different visions for the future of the technology.

In October 2011, Bio-IT World ran a cover feature on Toumazou, a microelectronic engineer who had made an unlikely turn into biotechnology in the middle of his career. At the time that story was published, Toumazou had spent nearly ten years working on semiconductor sequencing, first in his academic lab at Imperial College London and later at DNAe. After a decade perfecting a handheld microchip that could read short genetic sequences, Toumazou seemed about to strike it big. While Ion Torrent provided a major payoff by licensing his patents, DNAe itself was toying with a miniaturized device that could be used inside a doctor’s office or hospital; Toumazou was gearing up to release a series of pharmacogenetic tests on the platform, to help physicians tailor drug prescriptions to their patients’ genetic profiles.

By going after the most high-throughput uses of DNA technology, Toumazou believes, Ion Torrent has missed out on a key advantage of semiconductor sequencing: its ability to scale down. Because DNAe’s “Genalysis” platform has no optical sensors, there’s no bulky equipment to hold back miniaturization. A Genalysis device can be held between two fingers and used for fast, highly targeted sequencing.

“My heart was always in the point of care:

the speed, cost, scalability,” says Toumazou. “That was where I saw the real value of the semiconductor technology.”

And how about those pharmacogenetic tests?

“We had a couple of interesting programs, with Glaxo and with Pfizer,” he says. But gradually he came round to the idea that these partners were going to slow walk the technology, that industry wasn’t ready to limit its patient pool by targeting mass market drugs like blood thinners to specific subpopulations.

“Does big pharma really want this?” Toumazou asks rhetorically. “The number of times I’ve stood at personalized medicine conferences, and you can hear how much big pharma wants it — but do they really want it?”

SKIN DEEP As a matter of fact, there is one place you can use a DNAe product for targeted genotyping today. It’s a shop on Bond Street, the nexus of London’s high-end fashion world and, not coincidentally, the second most expensive real estate location in Europe.

The shop is called GENEU (pronounced gene-you). It looks a bit like an Apple store, and it sells skin cream. If you book an appointment, you’ll be greeted by a personal care assistant with a PhD, who collects your saliva using a cheek swab. Your DNA is then extracted from that swab and fed into a Genalysis device, which half an hour later will have concocted your personal U+ profile: a set of genetic variants that GENEU believes affect your skin health.

You leave the store with a skin cream in which ingredients have been blended

according to your unique DNA code. The business is apparently quite profitable.

GENEU’s service is not exactly an ennobling vision of the future of medicine, but, says Toumazou, he’s got his foot in the door. “You can take the stigma away from medical technology by presenting it in a luxury environment,” he says. “What you’re doing is getting the consumer to appreciate the value of personalization in an industry that doesn’t have the big pharma regulatory barriers.”

The trick for DNAe is to find a medical niche for the Genalysis platform where the need for a fast, highly portable device is obvious, and a larger whole genome sequencer would not get the job done. Pharmacogenetics made sense because doctors might want to run tests in their offices before prescribing a new drug, but the interests of big pharma aside, that field has had a hard time convincing the medical community that DNA is a better indicator of drug response than age, BMI, or traditional blood tests.

Toumazou is also interested in oncology, which is leading the way on connecting therapies to personal genetic information. Many next-generation cancer medicines are only effective against tumors with certain types of mutations, making it well worth physicians’ while to dip into their patients’ genomes. But here, DNAe might have trouble competing with whole genome sequencing. Cancer tests are rarely urgent, and the range of genes involved in choosing a therapy can be very wide, making it more practical to send specimens to a lab with an Illumina or Ion Torrent machine to sequence hundreds of genes at once. Plus, until someone figures out how to reliably extract tumor DNA from the bloodstream, getting tissue samples from patients will remain an invasive and time-consuming











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process, limiting the benefits of a fast turnaround for the test itself.

“DNAe [invented] a solution that was looking for a problem,” Toumazou admits. “We’re pretty open to all options. This is blood-to-result. It’s a diagnostic, really, so it’s a very different sell to a sequencing machine.”

THE GERM OF AN IDEA When would you need to sequence a small amount of DNA in a hurry? For clinicians dealing with infectious disease, the answer is all the time.

Bacterial infections can be tricky to diagnose, because their symptoms are often non-specific and it takes time to grow cultures in the lab or run a series of one-off tests to confirm suspected diagnoses. Meanwhile, an infection that progresses to sepsis can become life-threatening in a matter of hours. With a platform like Genalysis, even a small amount of genetic information could be used to confirm the species and strain of an infectious agent, as well as flag mutations that give rise to antibiotic resistance or virulence factors.

The DNAe team has always known that bacteria — and viruses and fungi — were promising targets for their technology. Splitting samples into different chambers on the Genalysis chip, they could run multiplexed genotyping tests for a variety of infectious agents at once. Alternatively, they could perform targeted sequencing of gene regions broadly shared across different species. “We’ll be looking at very wide-ranging primers that allow us to sequence within a particular targeted range,” says Toumazou. “It’s so versatile, because you can amplify, you can genotype, you can hybridize, and you can sequence all on the same chip.”

The obstacle has been bringing samples to the platform. There’s only so much advantage to be had in quickly sequencing a pathogen’s DNA on a microchip if users still need a full suite of lab equipment to isolate the DNA in the first place. But with infectious disease looking more and more like DNAe’s best point of entry to the clinic, the company is now pressing ahead with pathogens. Toumazou successfully raised enough funding in a venture round last April to scale up operations, and earlier this year, DNAe paid around $24 million to acquire nanoMR, a small company based in New Mexico with a product that quickly extracts bacterial and fungal cells from blood.

“One of the USPs [unique selling points]

of our technology has always been its miniaturization capability and its speed,” says Toumazou. “So we were looking for a front end that could be scaled in a very similar way.” nanoMR’s Pathogen Capture System is a portable platform that works by attaching tiny magnetic beads to antigens, which bind with antibodies on the surface of their target cells. The beads can be used to grab pathogens from whole blood, and then magnetically separated from the blood sample.

Toumazou plans to combine the Pathogen Capture System with the Genalysis platform to build a single instrument that goes straight from sample to answer. “It will be a cartridge-based system,” he says, “a bit like a USB stick, a portable unit that you

“DNAe [invented] a solution that was looking for a problem. We’re pretty open to all options. This is blood-to-result. It’s a diagnostic, really, so it’s a very different sell to a sequencing machine.”

Chris Toumazou, CEO of DNA Electronics











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plug in.” DNAe’s software will also cover interpretation of the results, so physicians who run tests on the instrument can get back a diagnosis and recommended drug courses.

CHIPS ON THE TABLE Bringing a product like this to the clinic, where it would be used to make life-or-death decisions for vulnerable patients, is a harder proposition than bespoke skin care. But Toumazou is not the only one who sees targeted sequencing as the future of infectious disease diagnostics. Illumina and Ion Torrent sequencers are already being used to monitor bacterial outbreaks in real time, and it would be a short (though sensitive) leap from there to diagnostic tests. A newer handheld sequencer made by Oxford Nanopore in Toumazou’s native England is also showing some early promise in this area. And regulators are slowly warming up to the idea of using new DNA technologies in the clinic.

“The FDA are getting a lot more relaxed on this,” says Toumazou, citing companies like BioFire and T2 Biosystems, who both have FDA-cleared platforms that use DNA signatures to identify pathogens. (For a profile of an instrument along these lines, see Bio-IT World’s feature on QuantuMDx.) These companies’ tests are more specific than the targeted sequencing Toumazou

wants to do at DNAe, but they do suggest that genotyping, at least, is within the FDA’s comfort zone.

“We know that we’ve got this wonderful ability to scale semiconductor chips, so you can highly target,” he says. “I think that semiconductors will play a very important role in this next decade, where you’ve got optics that don’t scale down to the molecular level or the chip level, and you’ve got nanopore that doesn’t scale up to the level where you can get any accuracy.”

Toumazou’s excitement for semiconductor sequencing has not diminished in the years since Ion Torrent first brought his technology to light. If anything, he’s ready to raise the stakes.

“This has to be global,” he says, noting that point-of-care tests for infectious disease

are most urgently needed in low-income countries with little access to hospitals. “Southeast Asia, Africa, these are the places we really need to target. This will really test the technology to its extremities.”

For a platform currently serving the luxury fashion market, it’s a tall order to develop a product that could be deployed in India while clearing regulators in the U.S. and Europe. But Toumazou’s unwavering optimism is not out of place for a sequencing technologist, a breed of inventors that has gotten used to shattering new barriers year after year. And even as mainstream genomics has continued to trend toward bigger, more elaborate sequencing projects, the thrill of reading short strings of DNA with a chip the size of a postage stamp has not worn off. As DNAe turns once more to new horizons, there’s still reason to think Genalysis has not yet had its chance to shine.

“This has to be global,” he says, noting that point-of-care tests for infectious disease are most urgently needed in low-income countries with little access to hospitals. “Southeast Asia, Africa, these are the places we really need to target. This will really test the technology to its extremities.”

Chris Toumazou, CEO of DNA Electronics











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PacBio just released the world’s best whole genome sequencer. Will it be enough to keep the company afloat?

BY AARON KROL | OCTOBER 1, 2015

This Wednesday, in a surprise announcement, Pacific Biosciences of Menlo Park, Calif., confirmed rumors that it has been working on a smaller, more price-effective version of its RS II gene sequencer. But rather than push out a scaled-down benchtop instrument for simple use cases, as many had anticipated, the company unveiled a machine that improves on the RS II in every particular: less than half the cost, a third the size, and most importantly, almost seven times as powerful.

A WORTHY SEQUEL:PACBIO’S NEW SEQUENCING SYSTEM











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The new instrument is called the Sequel System, and PacBio expects to ship its first ten before the end of the year.

2015 has been a big year for new sequencing instruments, with every company that actually has a functional gene sequencer bringing something new to market. Illumina added the HiSeq 3000 and 4000, filling some gaps in its catalogue between the gargantuan HiSeq X Ten and the well-loved HiSeq 2500. BGI, which had previously offered its Complete Genomics technology only as a service, introduced the Revolocity system. Thermo Fisher refocused on what it does best, producing an Ion S5 series that will appeal to labs running high volumes of targeted gene panels. And while the idiosyncratic Oxford Nanopore Technologies has shrugged off the idea of traditional market launches, it did open an access program for its first high-throughput instrument, the PromethION.

Every one of these instruments (with the possible exception of the Revolocity) can reasonably claim to be the best at something. PacBio’s new Sequel System can’t compete on data volume with the HiSeq line or Complete Genomics, and won’t come close to the Ion S5 or Illumina MiSeq on price. It doesn’t have the wow factor of the Oxford Nanopore MinION’s extreme miniaturization.

But for the gold standard of high-quality, whole human genomes, PacBio has just

released the best sequencer money can buy.

The reason I feel comfortable making that claim without seeing any data from users — indeed, without any specific data from PacBio — is that the company already had the best sequencer for whole genomes, in the RS II. Based on the same SMRT (single molecule, real time) technology, the Sequel will have all the same advantages: a random error profile that provides very high consensus accuracy; built-in DNA methylation data; and extremely long reads that let users assemble entire genomes from scratch, revealing the large structural changes to the genome that get washed out on other sequencing platforms.

PacBio’s problem has always been cost. The RS II sold for $750,000, in the same range as the much higher-volume HiSeq series. A deeply sequenced whole human genome on the RS II would run to tens of thousands more in consumables. The Sequel, however, will be priced at $350,000, a little closer to a mid-range instrument like the Illumina NextSeq; and while its consumables will actually cost more per run than the RS II, the much greater volume of data generated more than makes up the difference. In a call to investors this morning, PacBio CEO Mike Hunkapiller said that the Sequel should deliver a 10x human genome in one day, at a consumables cost of $3,000. A really high-quality 30 or 50x genome would be proportionally more.

That’s more than five times the speed of the RS II, for less than half the cost. PacBio is still far from the cheapest option for labs looking to sequence genetic material, but it’s no longer a wild outlier, a fact that should let its technology’s inherent advantages shine.

PRICE PREMIUMPacBio, however, is still not in an enviable position. The company has always struggled to turn a profit. For investors, a key point about the Sequel will be that it can be manufactured for a quarter the cost of the RS II, giving PacBio its first really strong profit margin. But even the Sequel won’t escape the trap of being a niche product, for users who want to study structural variation, run complete de novo assemblies, or look into a few developing fields like epigenetics and alternative splicing of RNA. At best, the Sequel changes the calculation for lab heads who might have liked to lean in these directions, but have balked at the cost.

The PacBio Sequel System, still a full-size instrument but no longer the behemoth RS II. Image credit: Pacific Biosciences

The Sequel should deliver a 10x human genome in one day, at a consumables cost of $3,000.











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It feels unfair that PacBio has been left at the margins of the booming genomics industry. Like Ion Torrent (now owned by Thermo Fisher), PacBio overpromised in its early days and eventually had to accept that it was not catching up with the dominant Illumina on raw sequencing power, forcing a pivot to narrower indications where it could compete. But unlike Thermo Fisher, for PacBio that process involved calling out real and fundamental problems with the direction of genomic science. The deluge of fast, cheap, short-read technology had blinded the field to whole categories of genetic variation. Even if only out of sheer self-interest, the PacBio team has done as much as anyone to turn that around.

Long-read sequencing on the RS II has moved quickly over the past year, from a first whole human genome assembly in November 2014, to a pipeline today that can produce them on demand. Along the way, PacBio has fostered a body of software tools adapted to long reads and de novo assembly, and helped create a market for other companies like BioNano, RainDance, and 10X Genomics to build instruments that illuminate the structural complexities of the genome. This renewed interest in seeing the entire genome clearly is good for science as a whole.

The Sequel might finally let PacBio reap the profits from this field it’s helped to sow. The new instrument, backed by the same sample preparation and software pipelines as the RS II, will be immediately supported for all the same applications as its predecessor, giving this project maximum reach for any scientists who could be won over by PacBio’s new, much more attractive price point.

Nonetheless, there are still serious headwinds. Running a Sequel at scale remains a costly option, a sort of luxury genomics. What’s more, PacBio has genuine competition these days. Clever workarounds like 10X Genomics’ GemCode Platform make it possible to get something very like long reads out of an Illumina machine.

And that’s nothing next to what’s coming from Oxford Nanopore. No one knows if the company’s radically new technology will overcome all its accuracy issues, but experiments with the handheld MinION sequencer have produced some seriously cool results (often with help from software tools originally written for PacBio reads). The PromethION, essentially a battery of 48 MinIONs rigged together, promises to ramp the MinION’s ultra-low-cost long-read data up to the level of human genome sequencing — and if it’s successful, the technology has plenty of room to keep scaling up.

In short, the Sequel breaks new ground on the accessibility of true whole human genome sequencing, but it may not be on top for long.

A TOE IN THE CLINICStill, PacBio has a few tricks up its sleeve. Unlike the RS II, Hunkapiller told investors today, the Sequel is built with a flexible architecture that will let the company keep designing new SMRT cells with more waveguides, the rate-limiting structures for sequencing. Not only does the Sequel jump to one million waveguides, compared to the RS II’s 150,000, but users can now expect to see that number rise. In the fast-advancing world of genomics, that ability to keep scaling is crucial.

Perhaps even more importantly, PacBio is on track to become a force in diagnostics, a huge market that has barely been touched by sequencing companies. The Sequel is the fruit of a diagnostics-focused partnership with Roche, whose dogged determination to be a part of the sequencing revolution is unmatched anywhere else in pharma.

Roche’s efforts in genomics have been a bit hapless to date — the company has notably fumbled an attempt to buy Illumina, shuttered its subsidiary 454 Life Sciences, and abandoned a nanopore sequencing project — but with the Sequel, Roche has helped to add real value to its partner’s technology. Now, Dan Zabrowski, head of Roche’s sequencing unit, says that the Sequel will become the basis for a series of clinical launches beginning in late 2016, including an instrument that will aim for FDA clearance.

Neither partner is sharing what indications that program will focus on, but Huntington’s disease, fragile X syndrome, and a few other rare genetic conditions caused by structural variants are a good bet. And then there’s cancer, where there’s a huge need for a clinical instrument that can illuminate the large, compound mutations that in many cases drive tumors out of control.

These are all areas that, at least in the research lab, PacBio instruments have helped shed a great deal of light on. Whether the company will get a chance to bask in that light depends in large part on whether Roche can secure the first mover advantage in the clinic — and who’s coming up behind.











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THERMO FISHER CLARIFIES ITS VISION FOR SEQUENCINGWITH RELEASE OF ION S5 INSTRUMENTSBY BIO-IT WORLD STAFF | SEPTEMBER 1, 2015

T hermo Fisher Scientific, which owns the Ion Torrent line of next-generation sequencing instruments, announced today that two new sequencers are being added to its portfolio. The Ion S5 and Ion S5 XL will make up a new mid-range in the Ion Torrent series, with a price and throughput intermediate between the

cut-rate Ion PGM and the workhorse Ion Proton.

The release, however, is about more than filling a gap in Thermo Fisher’s catalogue. The Ion Torrent sequencers, built on a unique technology that measures the electrical output of DNA binding events on a semiconductor, were originally conceived as a way to cheaply read massive and

indiscriminate stretches of DNA, up to whole human genomes. The instruments are capable of this kind of sequencing, but since their introduction in 2010, their efficiency has been leapfrogged by Illumina’s HiSeq series, now overwhelmingly the go-to system for broad genomics projects.

On the other hand, users of the Ion PGM and Ion Proton have preferred this technology for targeted sequencing, using both marketed and custom panels to quickly cover selected gene regions. Thermo Fisher’s cancer panels have been deployed for choosing targeted therapies in oncology, and in basic research, Ion Torrent devices have powered facilities like the Mount Sinai genomics lab in Branford, Conn., where a 26,000-gene panel is being used to cover a spectrum of disease-related variants.











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“Targeted resequencing is the area we’ve had particular success in, especially for oncology,” Andy Felton, Thermo Fisher’s head of product management for the Ion Torrent line, acknowledged in an interview with Bio-IT World this March (unrelated to the S5 releases). “Our realization over the last year has been that we need to focus on that more ― not that we’re giving up on the exome and genome space, but we want to focus where our strength is and deliver products that really enhance our workflow.”

The Ion S5 and S5 XL are strong fits for that niche. Their sequencing specs are competitive for the price: depending on the chips used, S5 instruments can deliver up to 20 million reads of 400 base pairs, or 80 million reads of 200 base pairs, in each 2.5-hour sequencing run. That’s roughly three times the throughput of an Ion PGM, making the S5 a rapid alternative for the same targeted sequencing applications, while outperforming Illumina’s low-cost

MiSeq on volume and quality of data.

In addition, the instruments should be easier to use than previous Ion Torrent devices, an important consideration for the kinds of smaller academic and hospital labs that are most likely to work with targeted panels. The reagents to run the S5 series have been consolidated onto disposable chips, and on-board computing handles the first steps of analysis, a longer process than the sequencing itself. (The amount of computing power is the major difference between the standard S5 and the XL model.) The instruments can also be paired with the Ion Chef device for automated preparation of DNA samples, which the company says will result in just 45 minutes of hands-on time and two pipetting steps from raw sample to sequence.

“We believe that the Ion S5 Systems deliver the best value of any benchtop instruments in the industry,” wrote Ion Torrent General Manager Mark Gardner

in a letter introducing the new products. “We built the Ion S5 Systems with process-controlled, easy to use workflows in mind, while retaining maximum application versatility, rendering the Ion S5 Systems an outstanding platform of choice for partners to develop their own assays.”

Meanwhile, Thermo Fisher insists that the P2 chip, a long-planned upgrade to boost the throughput of existing Ion Protons for whole-genome sequencing, is still in active development. Repeated delays, however, may have done irreparable damage to that product. The chip is now more than two years overdue, and even once delivered it will not come close to matching the throughput of Illumina’s most advanced HiSeqs. In light of the major market changes of the past few years, introducing the S5 series is a crucial course correction for Ion Torrent, salvaging its sequencing technology for those applications where it is most competitive. Fortunately for Thermo Fisher, there is strong overlap between the S5’s strengths and the needs of clinical centers, where the market for sequencing is still young and very much up for grabs.

Taking this logic to its extremes, DNA Electronics, the company that originated semiconductor sequencing, is working on handheld chips for ultra-niche genetics, targeting just a small handful of variants at a time. Although semiconductors have not come to dominate population-scale genomics as some predicted they would five years ago, the technology could still be a central part of sequencing’s adoption in routine medicine.

“We believe that the Ion S5 Systems deliver the best value of any benchtop instruments in the industry,” wrote Ion Torrent General Manager Mark Gardner in a letter introducing the new products. Image credit: Thermo Fisher











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BY BIO-IT WORLD STAFF | OCTOBER 28, 2015

At the International Conference on Genomics held over the weekend in Shenzhen, China’s public-private genomics agency BGI revealed a new sequencer to be made available exclusively to Chinese customers beginning in 2016. The BGISEQ-500 is the third sequencer to be introduced by BGI ― the China FDA cleared two of the company’s instruments for use in hospitals last year ― but the first to have been substantially designed inside China. Previously, BGI had released the BGISEQ-100, a retooled Ion Torrent device, and the BGISEQ-1000, largely similar to the machines built by Complete Genomics of Mountain View, Calif. (now BGI’s American subsidiary) for its sequencing-as-a-service business.

The BGISEQ-500 also uses a variation on the “DNA nanoballs” sequencing method invented by Complete Genomics, but it has been hugely reengineered. While Complete Genomics’ instruments are restricted to sequencing whole human genomes and exomes, the new instrument is much more flexible and will launch with capabilities in RNA sequencing (including in single cells), panels for non-invasive prenatal sequencing, and genotyping panels, among other applications. It also has a greatly reduced footprint, fitting neatly on a benchtop.

No user data is available, but based on the BGISEQ-500’s specifications, BGI clearly intends for the sequencer to compete with

Illumina’s line of NextSeq instruments, as a benchtop machine with high-throughput capabilities. It is priced to undercut the NextSeq 500, at a 30-40% discount, and its reported throughput on the larger of its two flow cell options is 200 gigabases per run, roughly double NextSeq’s most powerful mode. The BGISEQ-500 will also be capable of rapid runs producing as little as 8 gigabases of data, an advantage for clinical assays; BGI plans to submit the instrument to the China FDA for clearance.

If these figures pan out in practice, Illumina could find itself in the unfamiliar position of competing on features like read length rather than on scale and price ― especially if the BGISEQ-500 is eventually released to global markets.

The instrument is the culmination of a long process that began with the purchase of Complete Genomics in 2013. At the time, Complete Genomics sequencing was available only as a service; BGI, already a much larger service provider in its own right, pivoted its new subsidiary entirely to making commercial instruments.

Complete Genomics was not, however, stripped for intellectual property; instead it has focused on its own sequencing system, Revolocity, unveiled this summer and also scheduled for its first installations in early 2016. Revolocity hews much closer to the original vision of Complete Genomics, to produce whole human genomes at an industrial scale. Under BGI’s influence, it has been redesigned for extreme ease of use in a clinical setting, with automation and software built in to go all the way from a blood sample to a variant call file.

BGI RETOOLS COMPLETE GENOMICS TECHNOLOGYFOR ITS NEW HIGH-THROUGHPUT BENCHTOP SEQUENCER











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BY AARON KROL | NOVEMBER 9, 2015

QIAGEN’s GeneReader DNA sequencing system was finally unveiled last week in Austin, Tex., at the annual meeting of the Association for Molecular Pathology. The company had first planned to launch the GeneReader in 2014, but ran into delays during early access testing.

QIAGEN, an all-around molecular diagnostics company with a large customer base in both clinical and research, has been planning an entry into next-generation sequencing (NGS) since at least 2012, when it acquired Intelligent Biosystems, a small genomics player from Waltham, Mass. QIAGEN has also picked up CLC bio and Ingenuity, two popular bioinformatics vendors, to build a software suite alongside its sequencing system.

QIAGEN is making a late entry into NGS, at a time when even better-established vendors, like Thermo Fisher and Pacific Biosciences, are fighting to hold onto a meaningful share of a market dominated by Illumina of San Diego. But QIAGEN is not the only company that believes a huge, untapped base of hospital labs will soon be using sequencers as part of regular patient care, providing a chance for new technologies to get a foothold. In principle, QIAGEN’s existing relationships with these labs as a supplier of tests, reagents, and

equipment could help the GeneReader get traction, although if labs prefer to buy sequencing equipment from their reagent vendors, that hasn’t been obvious to date. (Just ask Thermo Fisher or Roche.)QIAGEN’s big pitch for the GeneReader is that users will not have to homebrew solutions for working with DNA samples or making sense of genetic data. The GeneReader will only be sold as a package with two other instruments, the QIAcube for extracting DNA from blood and

tissue samples, and the QIAcube NGS, which prepares those DNA libraries for sequencing. The system also comes with QIAGEN Clinical Insight (QCI), a platform that combines tools from CLC bio and Ingenuity, to analyze the raw data from the GeneReader and report on the clinical meaning of any genetic variants found.

“Labs struggle with the adoption of NGS, and we feel that QIAGEN is uniquely positioned to help them with those

The complete GeneReader system, including QIAcube, QIAcube NGS, and a computer running the QIAGEN Clinical Insight platform. Image credit: QIAGEN

QIAGEN RELEASES GENEREADER FOR CLINICAL SEQUENCING IN CANCERNote from Allison Proffitt










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barriers,” Jonathan Arnold, QIAGEN’s Senior Director of Marketing for NGS, tells Bio-IT World. “We’re launching a truly complete NGS solution, and that’s very different than what any other vendor has done.”

Well, sort of. Complete Genomics, a subsidiary of BGI, took a similar tack with its Revolocity sequencing system this summer, although Revolocity’s built-in software only goes as far as calling genetic variants, not interpreting them for physicians. But unlike the ultra-high-throughput, whole-genome-processing Revolocity, the GeneReader is clearly a diagnostic instrument, best suited to targeted DNA testing for clear clinical results. It launches alongside a 12-gene cancer test called the Actionable Insights Tumor Panel, which scans almost 800 mutation hotspots in genes like BRAF and EGFR, looking for variants that can be used to help choose therapies for cancer patients.

As a benchtop instrument, the GeneReader should fit neatly into the workflows of small to midsize labs that might otherwise pick up an Illumina NextSeq, or an Ion PGM or Ion S5 from Thermo Fisher, for targeted sequencing panels. QIAGEN is also offering a flexible pricing structure to win over labs that might want to run NGS, but don’t test at a high enough volume to justify buying a sequencer outright.

“We’re in these labs today,” says Arnold. “I’ve seen estimates that 75 to 80% of NGS labs are using a QIAGEN solution. And we have product lines, whether it’s qPCR or multiplex assays, that give us channels into these labs outside NGS.”

THE SPECSGenomics researchers and bioinformaticians want to know the specs of a new sequencer: how much data it produces per run, its error profile, its read

lengths. QIAGEN hates talking about the specs. The company line is that most of these metrics are irrelevant to a system that’s only meant to run panel tests, with the analysis and interpretation baked in.

“A lab does not need a bioinformatician to process this,” says Arnold.

Regarding volume, Arnold says the GeneReader system can run up to 120 panels per week. Other metrics are in service of those panels. For instance, the sequencer’s read lengths are around 100 base pairs, but not necessarily because of any technical limits. “We’re focused on somatic cancer, specifically from FFPE samples, so a read length of 100 base pairs is what that type of application needs,” Arnold says. “That’s what we designed around.”

Similarly, QIAGEN prefers to talk about accuracy in terms of test results, not base calls. At the Association for Molecular Pathology meeting, early access users from the Broad Institute of MIT and Harvard showed that the GeneReader, running the Actionable Insights Tumor Panel, picked up all the same mutations as an Illumina sequencer, and gave equivalent results to QIAGEN therascreen PCR tests.

Under the hood, the GeneReader runs on very familiar technology. The sequencing-by-synthesis method QIAGEN inherited from Intelligent Biosystems works the same way as Illumina’s machines, flooding the sample DNA with fluorescently labeled

nucleotides and imaging the results. (At one time Illumina and Intelligent Biosystems were involved in a series of lawsuits over this technology, although everyone’s intellectual property was left intact.)

The most unique feature of the GeneReader is that it can stagger samples. The sequencer reads up to four flow cells at a time, each with up to ten samples ― but if you start sequencing with the machine only partly full, you can add more flow cells mid-run. The GeneReader pulls this off by processing flow cells in “turntable” fashion, physically separating each sequencing step: adding new nucleotides, imaging, cleaving the fluorescent markers. New flow cells just slot into place between steps.

The feature is aimed at clinicians, who may have to start new tests in a hurry and can’t always predict their volume.

By Arnold’s count, the GeneReader can process 5,000 panels a year, enough for even fairly high-volume labs. So the 20-flow-cell sequencer that Intelligent Biosystems was at one point designing is probably not forthcoming from QIAGEN. “The sequencer itself will grow with the lab as their NGS business and volume grow,” says Arnold.

A TESTING MACHINEQIAGEN would like customers to see the GeneReader less as a device for unraveling the DNA code, and more as a high-

“We’re focused on somatic cancer, specifically from FFPE samples, so a read length of 100 base pairs is what that type of application needs. That’s what we designed around.”

Jonathan Arnold, QIAGEN’s Senior Director of Marketing for NGSNote from Allison Proffitt










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throughput testing machine. (In that regard QIAGEN is a lot like Direct Genomics, the Shenzhen-based company whose GenoCare sequencer is in early test runs with three Chinese hospitals.)

Of course, the GeneReader is still a sequencer, and in theory users can do whatever they want with it, from running third-party panels to sequencing bacterial genomes. The embedded software will even help with interpretation, to some extent, for pretty well any use case in humans. The former Ingenuity platform ― now QCI Interpret ― finds and reports disease-causing variants across the human genome, although QIAGEN is careful not to make any claims for the clinical validity of findings outside its own panels.

“We’re very focused on our Actionable Insights Tumor Panel,” says Arnold. “We verify that panel’s performance all the way from the GeneReader’s FFPE kit to the backend bioinformatics and QCI Interpret.”

That panel covers much the same ground as QIAGEN’s existing line of therascreen PCR tests for cancer, but also ropes in some extra gene regions with links to drug labels, the scientific literature, and testing guidelines from major clinical organizations. It also comes with a nifty extra feature in QCI Interpret: information on any ongoing clinical trials connected to a patient’s cancer mutations, organized by zip code.

For the time being, the GeneReader’s “on-label” applications will stay firmly in somatic cancer. QIAGEN has made sure the instrument can work with the degraded DNA in FFPE (formalin-fixed, paraffin-embedded) samples, and is planning to launch a solution for liquid biopsy as well. The many other potential uses for NGS ― like infectious disease, prenatal testing, and rare disease ― are on the back burner.

QIAGEN is being coy about the cost of the GeneReader, which it expects to sell mostly on a “price-per-insight” model. “I can tell you we will be extremely price competitive with what’s out there today,” says Arnold. “We would sit down with a lab, talk about the number of samples they’re going to be running, and come up with a price based on these different parameters.”

It’s a smart strategy to expand the number of customers who could think about adopting NGS. Clinical labs that run sequencing panels today already have their technicians trained to prepare DNA libraries, and more importantly, have bioinformatics pipelines in place to deal with the data, either homebrewed or from a vendor. Most likely, they employ experts in genetic interpretation, who can design new tests and know how to deal with ambiguous results. It won’t be easy to win these labs over to a new sequencing system when they’ve already invested heavily in getting this expertise and equipment in-house.

A price-per-insight model lets QIAGEN widen the field, offering more clinics access to the kind of broad cancer testing they might now be farming out to companies like Foundation Medicine, and promising more applications to come. The GeneReader probably won’t steal any customers from Illumina, but it might make labs eyeing their first MiSeqDx think twice about their choice of vendors.

NEW REGULATORY FRONTIERSQIAGEN’s plan is to submit both the sequencer and the Actionable Insights Tumor Panel to the FDA for clearance, but until then, it’s selling both of them for research use only.

That means the company has to be a bit circumspect with how QCI Interpret reports findings to doctors. “We make no claims about [the Actionable Insights Tumor Panel] as a diagnostic tool,” Arnold says. “We’re making no diagnostic claims in the interpretive reports. We’re not guiding therapy selection. We’re providing the relevant variants, but nothing more than that.”

That could change if the GeneReader and its cancer panel eventually win FDA clearance. QIAGEN is testing the waters for a broad form of genetic testing, very different from the tightly-focused NGS assays, like Illumina’s tests for cystic fibrosis, that the FDA has cleared in the past. The Actionable Insights Tumor Panel is more like the kind of sweeping genetic testing that more advanced clinical labs have undertaken on their own initiative, under FDA exemptions for laboratory developed tests.

These types of panels already have wide buy-in from professional organizations like the Association for Molecular Pathology. And QIAGEN isn’t going out on a limb with its genetic targets, mainly testing genes that the FDA has already acknowledged are linked to treatment options. It would be a good sign for the future if QIAGEN, which knows the FDA better than any of its competitors in NGS, could begin to bring these more wide-ranging uses of sequencing under the normal regulatory umbrella.

Right now, the Actionable Insights Tumor Panel is in the odd position of being narrower than many tests already in use, but more expansive than anything the FDA has so far approved. The launch of the GeneReader will be yet another nudge to regulators to clarify where genetic testing in the U.S. stands, joining a new class of sequencers that are, more than ever, taking NGS to the bedside.











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DIRECT GENOMICS’ NEW CLINICAL SEQUENCER REVIVES A FORGOTTEN DNA TECHNOLOGY











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BY AARON KROL | OCTOBER 29, 2015

BGI ― formerly the Beijing Genomics Institute, China’s contribution to the Human Genome Project, and now a hybrid state agency and private corporation ― is one of the world’s largest scientific research and industrial powers. From its headquarters in Shenzhen and outposts across Asia, Europe and the United States, BGI performs population-scale genomics studies, runs the world’s largest on-demand DNA sequencing service, and sells a small but growing suite of commercial products. Last week, BGI revealed the first sequencing instrument to be developed and produced in China, the BGISEQ-500, launched exclusively to Chinese markets.






Like other recent Chinese accomplishments in high-tech fields, the sequencer is as much a point of national pride as it is a commercial venture. “Shenzhen has transformed itself from labor-intensive industry to high tech,” says He Jiankui, a specialist in genomics and biochemistry who teaches at the city’s South University of Science and Technology of China. “The government has ambitions. They’re trying to switch from ‘Made in China’ to ‘Invented in China.’”

The BGISEQ-500, in fact, is based on American technology, which BGI bought up with the California-based Complete Genomics in 2013. But reimagining that technology, formerly housed in refrigerator-sized sequencers and manned by a small squadron of lab techs, to work in an easy-to-use benchtop instrument was a major feat of engineering. With the enormous demand for DNA sequencing in Chinese hospitals, it could be a commercial coup as well.

It’s a feat that He would like to replicate. In addition to running his university lab, He is the founder of Direct Genomics, a small company that aims to be the second to bring a Chinese-made sequencer to market. Direct Genomics already has a prototype instrument, called the GenoCare Analyzer, up and running, and this week the company announced a partnership with three hospitals in Shenzhen and Guangzhou to pilot a limited set of clinical tests.

“We’re a new generation of entrepreneurs,” He tells Bio-IT World. “We’ve had great discussions with the Chinese FDA, and they’re very welcoming for us to apply for clearance. They really hope our Chinese brand could be used in hospitals.”

Like the BGISEQ-500, GenoCare is at heart built on an American sequencing company’s technology, given a significant overhaul in Shenzhen. But He didn’t have to buy a big

player like Complete Genomics to get his hands on the intellectual property. Direct Genomics licenses its IP at a bargain from Caltech, using a sequencing method first brought to market by Helicos Biosciences of Cambridge, Massachusetts. The technology has been out of production for five years, and Helicos itself went bankrupt in 2012.

Geneticists never took to Helicos ― the company sold only around a dozen sequencers in the brief period, from 2008 to 2010, when its HeliScope instruments were on the market. But He thinks a new generation of customers might see the technology differently.

REVIVING HELICOSThe story of Helicos is an interesting wrinkle in the history of DNA sequencing. The company was founded in 2004 by Stephen Quake, then a professor at Caltech, who had come up with a way to sequence single molecules of DNA without first copying them through polymerase chain reaction. No previous technology had attempted this; in theory, single-molecule sequencing could correct for PCR biases, work with smaller volumes of sample DNA, and offer unprecedented accuracy in any experiment that involved counting DNA molecules.

In practice, Helicos was never a frontrunner in the field. By the time the HeliScope reached customers, Illumina of San Diego was producing DNA data faster, more cheaply, and with higher accuracy. But Helicos technology did manage to sequence a whole human genome (Quake’s), and in its time, the HeliScope could run more sequencing reactions in parallel than any other instrument available.

“I was very disappointed at the demise of Helicos,” says Bill Efcavitch, the company’s former Chief Technology Officer, and now

A prototype GenoCare Analyzer with its hood removed. Image credit: Direct Genomics











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Chief Scientific Officer of a DNA synthesis business called Molecular Assemblies. “I don’t think the Helicos technology was finished evolving.”

Helicos did hang around just long enough for a particular post-doctoral fellow in Quake’s lab to get acquainted with the technology. He Jiankui, who had just gotten his PhD at Rice University, joined Quake in his new lab at Stanford for one year in 2011. It was a brief, fortuitous chance for He to learn the ins and outs of single-molecule sequencing ― as well as work with a scientist with a long track record of spinning off companies from his university research.

That was exactly what He planned to do after returning to China, although it would take him a couple of years to form a clear picture of his own company’s purpose. From 2012 to 2014, as He mulled over business ideas ― and most of the remaining HeliScopes were shipped to Woburn, Mass., for a tiny sequencing-as-a-service business ― the market for genomics went through a radical change. Hospital labs dramatically ramped up genetic tests for cancer mutations and rare hereditary diseases. In the U.S., Illumina became the first company cleared by the FDA to sell sequencers explicitly for clinical use. BGI would soon follow in China, where an especially large market appeared for non-invasive prenatal testing (NIPT), a method of sequencing blood samples from pregnant mothers to detect serious conditions like Down syndrome in their developing fetuses.

Finally, in 2014, He came up with his pitch: to revive the old Helicos technology, but with an extremely narrow form of sequencing in mind. Direct Genomics would build an instrument meant only for the burgeoning clinical market, devoted to highly targeted sequencing for diagnostic tests. He contacted Quake and Efcavitch,

along with his former advisor at Rice University, Michael Deem, to see if they would be interested in serving as scientific advisors to his company.

“The original Helicos was doing whole genome sequencing,” He says. “But now the major market for sequencing is not in research. It’s in clinical analysis, where whole genome sequencing is not popular.”

Quake and Efcavitch were eager to sign on, in part because He’s vision takes advantage of an aspect of Helicos sequencing that never caught fire in a research setting. Other sequencing methods require DNA samples to go through rounds of PCR, or chemical tweaks to the native DNA molecules ― a small burden for researchers, but a big deal for busy clinicians with limited lab training.

The Helicos method, however, reads raw, unmodified DNA. Sample preparation from a blood or tissue sample is just two steps: isolating the DNA, and shearing it into fragments. “The sample prep for this kind of single-molecule sequencing is by far the most straightforward of any technology out there,” says Efcavitch. “And that’s a key point for a clinical setting.”

At Direct Genomics, He has also worked out a way to bypass target enrichment ― an extra sample preparation step for targeted tests, to make sure only DNA from the gene regions you’re interested in testing gets into the sequencer. Helicos sequencing worked by anchoring sample DNA in place on a flow cell, with small single-stranded DNA fragments as the “anchors.” On the GenoCare instruments, He has simply redesigned the flow cells so that those anchors will only bind with specific DNA sequences, tailored to the needs of each test.

“For all other next-generation sequencing, the target enrichment and sequencing are separate things,” says He. “What we do is

combine the two steps together. In one flow cell we do the targeted capture and the sequencing.”

That means the flow cells aren’t multi-purpose like in a research-grade sequencer, but for an instrument designed for diagnostics, that doesn’t matter. Direct Genomics will release a series of different flow cells for different tests, and clinicians will use exactly the same sample preparation steps for each one.

REENGINEERINGIn the years since Helicos closed its doors, there have been several agnostic advances in technology that have let Direct Genomics build a more efficient machine than the old HeliScope. The GenoCare Analyzer has new optics, cameras and flow cells; it’s a fraction the size of a HeliScope (about as large as a file cabinet) and much less expensive. While GenoCare’s total data output is lower than a HeliScope, its runs are also much faster, sequencing almost ten times as many bases of DNA per hour.

Still, for almost any research purpose, GenoCare would be a truly poor choice of sequencer. Its throughput, about 10 gigabases of DNA per 15-hour run, lags far behind the competition. Worse, GenoCare reads DNA in fragments of 35 bases or less, a small fraction of the read length of other sequencers. For any geneticist trying to stitch together reads to create a larger picture of the genome, these tiny fragments are not only computationally demanding, but also completely obscure important deletions and rearrangements in the genetic code.

GenoCare also suffers from a particular weakness that Helicos never overcame. Its chemistry, in which fluorescently-labeled bases are added to the sample DNA one at a time and photographed, is vulnerable to











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“dark bases,” where the fluorescent label is lost before it can be imaged. That leads to a high rate of false deletions in the sequence, which are hard to correct with a single-molecule technology that doesn’t have the high redundancy of other methods.

To some extent, Direct Genomics is working to fix these problems; He says that the read lengths, for instance, can get as high as 200 bases if needed. But for the most part, the company is just dodging GenoCare’s faults as a sequencer, by picking clinical targets where its flaws are irrelevant.

Take cancer mutations, one of the first indications Direct Genomics is piloting with its hospital partners. A large body of mutations are known to occur frequently in cancer cases, several of which indicate a good chance that a particular therapy could be effective in attacking the tumor. These cancer signatures can often be identified by single-base differences from the wildtype genome, as in the case of a panel of eight mutations Direct Genomics hastested its technology on.

“For some cancer genes, we know the target, we know where a mutation could happen, and we design a probe that’s very close to that hot spot mutation,” says He. “Then we can sequence a very short fragment to get an answer.”

Designing panels that look for single-base substitutions, instead of deletions or insertions, also lets Direct Genomics play to its strengths. “Our substitution error rate is very low, half a percent,” He says. “So it meets our clinical use requirements.”

This isn’t an infinitely flexible approach. It will never offer users a chance to test for large structural rearrangements in cancer, for instance. But considered as a diagnostic device, GenoCare is hugely versatile, covering indications in cancer, infectious

disease, and hereditary conditions. Direct Genomics has already made panels for NIPT and to test for antiviral resistance in Hepatitis B, which affects well over 100 million people in China.

“An important strategy for Direct Genomics is the correct choice of applications here,” says Michael Deem, who has been traveling in China with He to help assess the market. “There’s something like 1,400 tier-one hospitals in China that are focused on cancer, so it’s a big market for cancer diagnostics and monitoring… The easy sample preparation is really addressing the cost, labor, and technical concerns to make this feasible in the clinic.”

THE CLINIC OR BUSTGenoCare isn’t the first sequencer built for clinical tests, but it’s the first to so thoroughly write off anything else you might do with genomic data.

If everything goes as planned, Direct Genomics customers will never see much of the underlying DNA data they’re generating: the company will write software for each test to analyze the sequencing signals and report back variants. “It will all be on the machine directly,” says Deem. “The flow cell will be designed for a panel, and then the software would be analyzing the results from the cell to get the answer for your panel test.”

In this sense, a GenoCare panel could feel less like a sequencing run and more like a DNA microarray, a multiplexed test for a specific set of variants. Microarrays, however, involve a great deal of hands-on work in the lab, treating your sample to isolate and label the DNA of interest. The GenoCare gives the same results just by putting raw, unselected DNA in the flow cell.

Direct Genomics plans to work with its first

hospital partners to put together a core set of tests for launch, and to make sure its performance is consistent on real clinical samples. (Internally, the company has mostly tested its technology on synthesized DNA, although it has performed NIPT from real blood samples.) These tests on live samples will be the first demonstration of some of He’s most optimistic claims about his technology ― like that GenoCare will be able to work with very small quantities of DNA, or with the highly fragmented and degraded DNA found in cancer biopsies.

Fitting GenoCare into the hospital workflow, once the company scales up from its prototype instruments, will be an even greater challenge. “The idea,” He says, “is if a patient comes into the hospital and gives blood in the morning, we could load the DNA into the sequencer in the early afternoon, and then the patient comes back the next day and gets a result.”

“That’s the goal for any diagnostic tool,” Efcavitch adds. “Physicians really don’t want to be burdened with sequencing information. They want a direct statement: here’s a mutation.”

It’s not often that a fast-paced field like genomics dips into technology that was once written off as obsolete. But in a strange way, Helicos ― too slow, too flawed and too expensive to compete seven years ago ― might have been a little ahead of its time. While no one had much use in 2008 for sequencing directly from raw DNA, large-scale NIPT and cancer testing is a new niche that has yet to settle on a single technology.

“Steve Quake and I periodically reminisce about what Helicos could have or should have been,” says Efcavitch. “I’m very optimistic, and I’m pleased to see the technology coming back. It’s a brand new market.”











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BY AARON KROL | JANUARY 12, 2016

The JP Morgan Healthcare Conference comes but once a year. Now it’s here, now it’s here, and Illumina CEO Jay Flatley has honored his seasonal tradition by releasing a new next-generation sequencer in his address to investors at the San Francisco event yesterday morning.

Illumina is not quite a monopoly power in DNA sequencing, and increasingly there are interesting reasons a lab manager might mull over instruments from competitors―like a PacBio Sequel for long DNA reads, a QIAGEN GeneReader for on-demand clinical tests, or an Oxford Nanopore MinION for the sheer sci-fi swagger of carrying a sequencer in your coat pocket. But at the nexus of price, accuracy, and volume, Illumina still sets the standard for what labs of all sizes can achieve with sequencing, so the company’s annual release of its pent-up creative energies at JP Morgan is an important yardstick for the state of genomics.

This year, Flatley has unveiled the MiniSeq, Illumina’s most affordable sequencer yet. The instrument is a natural evolution of the product line. Two years ago, Illumina came out with an ingenious (though not universally loved) new chemistry system that uses just

JOLLY JAY FLATLEY USHERS IN

ANOTHER BIG YEAR FOR ILLUMINA

Illumina still sets the standard for what labs of all sizes can achieve with sequencing, so the company’s annual release of its pent-up creative energies at JP Morgan is an important yardstick for the state of genomics.











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two dyes, in different combinations, to label all four DNA bases. This system let the company scale back from the bulky optics in its top-of-the-line HiSeq instruments, producing the NextSeq, a much smaller and less expensive machine that approaches the HiSeq’s speed and throughput.

The MiniSeq applies the same principle to the baby of the Illumina family, the benchtop MiSeq. Reengineering has not only worked in simplified optics, but also stripped out one of two valves and pump systems in the MiSeq. More importantly, Illumina has designed the MiniSeq to run with a single reagent cartridge, which can be shipped dry, making it the company’s easiest sequencer to start using. Library preparation is still a hurdle for first-time users, but once a DNA library is prepared for sequencing, it can simply be loaded onto the MiniSeq’s single flow cell, and inserted in the machine with a new reagent cartridge.

At just over 18 inches to a side, the MiniSeq is the smallest Illumina sequencer, and at $49,500, it’s half the price of a MiSeq―the first Illumina instrument to be priced in line with Thermo Fisher’s Ion PGM. Illumina says that runs on the MiniSeq will take 24 hours or less, depending on application, and paired-end read lengths will top out at 150 base pairs. With a high-output reagent kit, the instrument will be able to deliver a whole genome of a midsize bacterium at about 30x coverage―although a more likely application will be targeted gene panels, an area where Illumina sees heavier competition from Thermo Fisher.

PROJECT FIREFLYNifty as it is, the MiniSeq is more a story of good engineering than a major scientific advance. A small sequencer along these lines would have been a safe bet for Flatley’s 2016 product reveal: Illumina’s dominance of ultra-high-throughput sequencing is

basically absolute, and the company already had two-dye sequencing in its bag of tricks. (That said, Keith Robison of the Omics! Omics! blog deserves to crow a bit about his uncannily accurate prediction.)

But Flatley also broke with tradition this year, trumpeting a technology he doesn’t expect to release until 2017. (And mid-to-late 2017 at that: sorry girls and boys, but Jay Flatley might not bring you any big presents next JP Morgan Healthcare Conference.) The new product, tentatively named Firefly, will be Illumina’s first sequencer that doesn’t rely on optical readouts. Instead, sequencing will be done on complementary metal-oxide semiconductor (CMOS) chips, a technology Illumina has said precious little about since it bought up a related company, Avantome, almost eight years ago.

Semiconductor sequencing is a downright cool technology, and when Ion Torrent was preparing to introduce it to the market for the first time in 2010, many felt this new method of reading DNA might take over completely. Semiconductor chips are cheap to fabricate and highly scalable, and can register DNA binding events in real time.

The trouble is that electrical signals on these chips are binary. That makes sequencing, which has to differentiate between four different DNA bases, an awkward fit for the technology. Like Illumina, Ion Torrent (now owned by Thermo Fisher) uses sequencing-by-synthesis, adding new bases to the sample DNA and recording them as they attach. But while Illumina, with its fluorescent labels, can add all four bases to the mix at once and see which one incorporates, Ion Torrent has to add them one at a time and check whether they incorporate, slowing down the system.

Now, Flatley says, Illumina has worked out a “one-channel” way of identifying bases. This is a bit like the clever trick they pulled with the NextSeq and MiniSeq, encoding four bases in different combinations of two dyes―but how they’ve further reduced this to a binary signal, Flatley isn’t saying. However it works, apparently the Illumina lab has already demonstrated the technology on individual CMOS chips, with greater than 99% per-base accuracy. This, Flatley told investors during a Q&A session yesterday, is “a pretty monumental achievement, frankly.”

The next step will be to incorporate those chips onto an easy-to-use instrument.

Given the history of pre-announced sequencing product launches, everything Flatley said yesterday about Firefly should be taken with a grain of salt, the 2017 launch date very much included. But his ambition is to sell the system for under $30,000, which would include both a sequencer and a separate box for automated sample preparation. Each of these instruments will be roughly a 12-inch cube, and freed from the bulky and heat-generating optical systems of current Illumina sequencers, they will be stackable, so that labs can keep buying them without eating up their lab space.

The MiniSeq, Illumina’s latest and smallest sequencing instrument. Image credit: Illumina











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Firefly’s throughput will be lower than the MiniSeq’s, but runs will be faster: it will produce about as much data per hour as a MiniSeq on mid-output mode.

Flatley says he had no choice but to jump the gun on announcing Firefly―its development will involve a substantial investment in 2016 and big changes to Illumina’s supply chain and manufacturing networks, all of which he felt was likely to tip off observers to the project.

Illumina’s sidestep into semiconductor sequencing is a nice reminder that genomics is blessed with one of the world’s better monopolies. Yes, more serious competition in DNA sequencing would be good news for pretty much everyone but Illumina. But if one company is going to have its fingers in virtually every sequencing lab in the world, at least it’s not sitting back raking in rent on its reagents. Whether it’s because of pressure from nanopore sequencing nipping at Illumina’s heels, or just a testament to the scientific talent the company has assembled, the world leader in sequencing is still sprinting to keep ahead.

THE REST OF THE PUDDINGAs usual, Flatley also previewed a smattering of other developments from the many arms of his company. He introduced a new, 96-sample bead chip called Infinium XT for Illumina’s DNA microarray business―increasingly an afterthought in the next-generation sequencing era, but still extremely useful for large-scale genetic research on the cheap. (Another sign of microarrays’ continued relevance this week was the astonishing $1.3 billion sale of Affymetrix, scooped up by Thermo Fisher at a 50% premium over the share price.) Infinium XT is set to ship in the second half of this year.

Another exciting hardware project is a

partnership with Bio-Rad Laboratories to build a high-throughput device for single-cell sequencing. This is an area of huge scientific value, giving researchers a much finer-grained look at genetic activity in living tissue. But methods for isolating single cells, and preparing them for sequencing, are painstaking and highly specialized. The new partnership will merge a droplet partitioning system created by Bio-Rad with built-in sample prep from Illumina, giving users an automated instrument that will go from cell samples to sequencing-ready DNA libraries at a cost of around a dollar per cell.

Last year, Flatley’s address focused heavily on real-world applications, showing off the extent to which Illumina had evolved beyond being simply a hardware company. Illumina still wants to have at least some stake in most of the end uses for its machines: Flatley announced 2016 plans to launch a tumor sequencing panel for the NextSeq, a cancer RNA panel, and a CE-marked prenatal test in Europe. But the emphasis of his JP Morgan address this year was more on the kit than the clinic.

That probably reflects a growing willingness to spin out riskier businesses outside Illumina’s hardware core. Illumina launched its first independent company, Helix, in August, for the decidedly unproven business of consumer genetics. The notion is that customers will get large portions of their genomes sequenced for well below cost, then pay to access that data through a variety of third-party apps. Flatley was able to announce yesterday that Duke University and Good Start Genetics have signed on as app vendors, joining the Mayo Clinic and LabCorp―but whether ordinary people will want to dip into their genomes enough times to keep Helix in the black is very much an open question.

Meanwhile, Illumina took another big

swing over the weekend with the launch of GRAIL. The premise of that company, to test seemingly healthy people for free-floating genetic markers of cancer in the blood, is both enormously tempting and painfully difficult. There are other liquid biopsy companies out there, but none aiming for cancer prevention. (With the rather shaky exception of Pathway Genomics.)

GRAIL expects to sequence samples at 1000x coverage to uncover trace amounts of tumor DNA, something that requires Illumina’s full resources. As Flatley told investors yesterday, “When we demonstrated internally how deep you had to sequence… we realized our customers couldn’t do this economically for a long time.” It seems the cost of this service, if it can prove itself in the clinic, will only be manageable with a steep discount on sequencing.

As 2016 opens, we’re starting to see an interesting new pattern for Flatley’s genomics giant. Proven applications stay in-house with the sequencers that make them work, with the hope that Illumina will be the go-to vendor as clinical sequencing continues to grow. Meanwhile, the company will place big bets on longshot ideas―but only if it can insulate them a bit from the core business, and raise some of the funding from venture capital.

The many starry-eyed biotech entrepreneurs gathering this week in San Francisco are probably taking notes. In 2007, Illumina was a secondary player in microarrays that vaulted over Affymetrix by predicting its own technology would soon go out of style. Now, with a market cap of $2.4 billion, the onus is on Illumina to capitalize on its lead. Whether the company’s restless activity pays off or not, you can’t fault them for not trying.











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BY AARON KROL | FEBRUARY 29, 2016

NanoString Technologies, a Seattle company with a small but comfortable niche in automated genetic analysis, is preparing to make the leap into DNA and RNA sequencing. The company revealed its novel sequencing process in a poster at the Advances in Genome Biology and Technology meeting, held in Orlando, Fla. earlier this month.

NANOSTRING REVEALS

NOVEL SEQUENCING METHOD FOR CANCER ASSAYS











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NanoString’s proposed sequencer is closely related to its current line of nCounter instruments, which target short genetic sequences with optically barcoded hybridization probes. In an nCounter experiment, a set of probes designed to bind with a specific group of genetic elements―for example, segments of genes that are commonly overexpressed in cancer―is mixed with a DNA or RNA sample. Each probe carries a unique sequence of fluorescent tags, the optical barcodes that are imaged to learn which genetic targets are present in the sample, and in what numbers.

The new sequencing process, named Hyb and Seq, uses very similar probes. But instead of just spotting key genes and variants, a Hyb and Seq experiment can read huge volumes of barcodes to recover the complete sequence of a DNA molecule.

NanoString is not trying to sprint to the front of the pack in sequencing, a competitive field where the market leaders are increasingly able to produce whole human genomes cheaply and quickly. Hyb and Seq, by

contrast, can only be used for targeted gene panels―focused experiments where users already have some idea what they’re looking for. But as the market for DNA information widens, there is more room for technologies that fill interesting niches, and Hyb and Seq has several unique properties that could give it an edge for NanoString’s core applications in cancer testing.

“It’s not for whole genomes,” says Joe Beechem, Senior Vice President of Research and Development. “It’s very much a targeted cancer panel we’re developing.”

In a Hyb and Seq experiment, single-stranded sample DNA molecules are immobilized on a flowcell and inserted into the instrument. (For its pilot studies, NanoString is using a modified nCounter SPRINT instrument, with added 3D-printed components.) The DNA does not have to be amplified or prepared with enzymes―the sequencing process works directly with the native molecules. Once the DNA is on the flowcell, a mix of over 4,000 different probes is added, at low concentrations that ensure probes will attach to the DNA more or less one at a time.

The probes in Hyb and Seq are much shorter than in NanoString’s gene expression assays, targeting sequences of just six DNA letters; together, they cover every possible six-letter combination of the DNA bases A, T, C, and G. After a probe hybridizes to the sample DNA, the sequence it targets can be read optically. The barcodes in Hyb and Seq consist of four fluorescent molecules, one for each DNA base, which attach sequentially to the probe and are imaged one at a time.

Reading the barcode reveals only that

a certain six-base sequence appears somewhere on the target molecule. On its own, this is a very small amount of information: the instrument doesn’t know where on the molecule this sequence occurs, and six bases isn’t enough to say much about what region of the genome a DNA fragment comes from. But the optics and chemistry cycle very quickly, so in a short time, the system picks up a lot of random six-base sequences. Because Hyb and Seq is used for targeted assays, with only a limited number of gene regions represented in the sample, NanoString’s software can rapidly eliminate regions that don’t have the right combinations of six-base sequences in them.

“You quickly know what gene you’re on,” says Beechem. At that point, additional sequence fills in gaps and covers genetic variants. “Then it’s just a matter of covering all the areas you want to sequence… It’s like a mini-assembly of the individual genes in your panel.”

“A HIGHLY ACCURATE READOUT VERY QUICKLY”Hyb and Seq will never be able to take on tasks like de novo sequencing, but for assays covering a few key genes, it has some intriguing advantages.

“It’s not for whole genomes. It’s very much a targeted cancer panel we’re developing.”Joe Beechem, Senior Vice President of Research and Development











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First, there’s sample prep. “It’s almost zero,” says Brad Gray, President and CEO of NanoString. Most sequencing technologies require sample DNA to be copied through the time-consuming process of PCR, producing huge volumes of DNA fragments to read in parallel. But Hyb and Seq looks at single molecules, so the only prep involved is to pull targeted gene regions out of the sample―in the case of cancer assays, typically DNA from a formalin-fixed, paraffin-embedded (FFPE) tissue biopsy. It takes just a few minutes to mix sample DNA with targeted capture probes and insert it into a NanoString flowcell for sequencing.

“The fact is that [sequencing] is still not a clinically friendly technology for a lab tech to run,” says Beechem. “There is still an unmet need for a radically simplified workflow and hands-on time.”

Hyb and Seq also has the unique property that the sample DNA is not damaged or altered. Barcoded probes can be repeatedly attached and washed off, so getting higher coverage to correct errors is as simple as resequencing the same DNA molecule over and over again. Beechem calls it “sequence until,” because the system can be programmed to keep running until it

reaches a certain accuracy threshold, rather than for a specified

number of cycles.

This could be especially useful when working with FFPE samples, where DNA is typically scarce and badly damaged. NanoString is designing its first demonstrations of Hyb and Seq for exactly this type of sample, which usually yields DNA fragments no more than three or four hundred bases long. “It’s a very natural extension of the business we’re already in,” says Gray. “The company mission is built around getting biomarker information out of tumor tissue, which can be used for research and ultimately in the clinic.”

Hyb and Seq is far from commercialization―Gray says a market-ready version, with a dedicated instrument, isn’t in the cards until at least 2017. But the earliest test runs suggest the system is workable, at least for sequencing small genetic variants on short DNA fragments. The rate of miscalled bases in each read is less than 3%, and while NanoString is still reviewing the data it has, Beechem says that so far there’s no indication of biased errors that would prevent that rate from improving with resequencing.

“Those are the most accurate single-molecule, single-pass error rates that have ever been measured, by a large factor,” he says. “And the fact that we can reread [DNA molecules] gives you a highly accurate readout very quickly.”

For all this early promise, NanoString is planning to enter one of the most competitive parts of the sequencing market. Cancer genomics is one of the few applications of DNA sequencing that can be used meaningfully in the clinic

today, and companies are hurrying to launch comprehensive assays that

small hospital labs can run in-house. Illumina’s line of TruSeq tests, and

Thermo Fisher’s AmpliSeq panels, cover much the same ground as NanoString’s proposed assays. And QIAGEN recently released the GeneReader specifically for the cancer market, running nothing but targeted gene panels. (Unlike with Hyb and Seq, this is a deliberate choice and not a technical limitation.)

Hyb and Seq will be fascinating to molecular biologists for its distinctive approach to getting sequence data, but whether it’s a leap forward for NanoString will depend more on mundane factors like speed and cost. Still, the minimum of hands-on time is a novelty that other technologies will have a very hard time matching, and the single-molecule process lends itself to tumor biopsies in a way that the competition does not. For now, this latest entrant into sequencing is a curiosity, but it’s a curiosity well worth keeping an eye on.

“The company mission is built around getting biomarker information out of tumor tissue, which can be used for research and ultimately in the clinic.”Brad Gray, President and CEO of NanoString











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