Illumina Unveils New Fleet Of Sequencing Instruments
By Deborah Borfitz
October 18, 2022 | The recent Illumina Genomics Forum occasioned the launch of several game-changing products, including the first FDA-regulated and CE-marked in vitro diagnostic (IVD) high-throughput sequencer (NovaSeq 6000 Dx) and the company’s most powerful sequencer to date (NovaSeq X series) that can run 20,000 whole human genomes a year on a single instrument. “NovaSeq X is our most sustainable high-throughput sequencer, enabling big science to be green science ... [in a year] already on track to be one of the top 20 hottest years on record,” according to CEO Francis deSouza during Illumina’s Innovation Roadmap session.
Illumina’s innovative sequencing technology, in the hands of brilliant minds, has already led to life-changing science, says deSouza. With the creation of HiSeq X in 2014, breaking the $1,000 barrier for human whole-genome sequencing, more genomes were sequenced in a single year than all previous years combined. The 2017 introduction of the company’s popular NovaSeq 6000 machine also helped enable comprehensive genomic profiling from liquid biopsies.
“Together, we’ve generated more data [more than one billion gigabases] than all other platforms combined ... the equivalent of six genomes every minute,” deSouza says. Illumina technologies have helped reduce sequencing costs and data complexity, allowing clinical genomics to move into regulated markets. Nearly four million people have now had their genome sequenced and thousands of tests based on next-generation sequencing have emerged, he notes.
“But even with this progress, we have barely scratched the surface,” continues deSouza. Current genomic knowledge is based on sequencing of less than 1% of all species, 0.07% of humans, and 0.0000001% of microbes. “We are at an inflection point today unlike any other in the history of genomics.”
NovaSeq 6000 Dx delivers what it takes to unlock the full power of the genome and make new breakthrough discoveries, namely “insane amounts of data, an incredible technology, and immense scale,” deSouza says. “We are pushing the boundaries of clinical testing with scalable, flexible throughput, and improved economics, and we are making it accessible for the 1.5 billion people who live in countries requiring regulated IVD products.”
The technology offers an open platform with RUO [research use only] and IVD modes and a dedicated DRAGEN server, he shares. Users get flexible, high-quality, automated data processing to reduce their hands-on time with a quarter of the touchpoints. Illumina’s priority now is menu expansion in collaboration with its IVD partners “to bring the highest standards of compliance and actionable insights for patient care,” adds deSouza.
Separately, Illumina Complete Long-Read technology (formerly known as Infinity) with DRAGEN analysis demonstrated its superior performance in terms of delivering the most accurate view of the genome when tested against benchmarking data sets from the PrecisionFDA Truth Challenge, deSouza points out. “But that is not good enough.” The company will now “close key gaps” on the remaining 5% of genetic regions that are harder to map to offer “a complete and accurate representation of the genome at the single molecule level.”
In 2023, Illumina will deliver two Long-Read products “with speed, scale, and low DNA input requirements,” he reports. Scheduled for launch in the first quarter is a whole-genome assay that uses long contiguous reads to extend the coverage and completeness of the 30x genome of NovaSeq. Illumina Complete Long-Reads is already “transforming lives around the world,” says deSouza, referencing its use in the discovery of rare and ultra-rare variants by Robert Fulton (assistant professor of genetics at Washington University Medical School) and Tracy Cole (senior director of research at n-Lorem).
In the second half of next year, Illumina will unveil an enrichment panel for Illumina Complete Long-Reads, targeting only difficult-to-map regions of the genome, to deliver “unrivaled throughput at the lowest incremental cost ... [and] the most comprehensive view of the genome at scale on a single platform.”
The NovaSeq X
Illumina’s XLEAP-SBS Chemistry (formerly Chemistry X) is the industry’s most widely adopted sequencing by synthesis chemistry, says deSouza, with a record-holding 300,000 scientific publications. “But we knew we needed more [and] that’s why we used the world’s largest proprietary pipeline of SBS technology development to engineer a new benchmark in sequencing chemistry.”
XLEAP-SBS is engineered for speed and greater fidelity with two times faster incorporation speed, up to three times more accuracy, and the potential for much longer reads, he continues. It will be available on Illumina’s flagship benchtop platforms NextSeq 1000/2000 in early 2024 and provide increased data quality even at higher flow cell densities.
With the addition of XLEAP-SBS chemistry and P4 flow cells, the platforms are expected to generate outputs of more than 500 gigabases, says deSouza. “These innovations will expand application breadth and drive sequencing costs down even more.”
But Illumina isn’t stopping there, says deSouza in introducing the NovaSeq X series, because “the insane amount of data requires something way more powerful.” The company is positioning the next-generation sequencer as the exemplar of the genome era when the dream of finding a cure for cancer and treatments for heart disease and diabetes could become reality.
Both the new NovaSeq X and NovaSeq X Plus configurations are powered by XLEAP-SBS and give “massive improvements in all key aspects—throughput, scale, accuracy, simplicity, efficiency, and cost, and we will do it more sustainably than ever,” says deSouza. The Plus version is capable of fully independent dual flow cell processing, delivering 16 terabases of output.
The NovaSeq platforms feature three types of flow cells with up to eight individually addressable lanes, and up to 16 per run, with automated and integrated secondary analysis capabilities to remove the bioinformatics bottlenecks, deSouza explains. NovaSeq X Plus has the power to generate 20,000 whole human genomes per year, which represents a two and a half times increase in throughput to support larger projects and deeper sequencing that is faster than ever.
A single box contains all the consumables, compared to three boxes required by NovaSeq 6000, he points out. “We have reduced packaging weight and waste, each by 90%. We’ve also reduced cartridge volume by 50% ... saving [freezer] space, labor, and money, and we reduced plastic mass by 50% while thermostable reagents provide ambient-temperature shipping—a genomic gamechanger.”
That means no more ice packs or dry ice, making high throughput sequencing more accessible than ever to countries that don’t have cold chains, says deSouza. “These groundbreaking green innovations not only help save the planet; they also save you time ... unboxing and discarding packaging waste.”
Scientists, engineers, designers, and developers at Illumina spent a lot of time optimizing virtually every aspect of the workflow so NovaSeq X is easy to use with the fewest possible clicks, he continues. The machines automatically populate planned runs so users can just select those they want and provide information on the runs so “it is really hard to make a mistake.”
The NovaSeq X platform has the power of three NovaSeq 6000s and a DRAGEN server—all in the footprint of the NovaSeq 6000, says deSouza. Its new, smaller flow cell, which is roughly the size of a cell phone, also has much higher output.
All the redesign work translates into maximum productivity and turnaround time for users, and makes the most of their precious samples, deSouza says. Collectively, these innovations deliver significant operational savings.
But the economic improvements don’t stop there, he adds. “You can now sequence at a list price of $2 per gene or $200 per genome ... [including] the cost of secondary analysis using the DRAGEN on board. NovaSeq X will foster a new wave of applications that require more sequencing per sample and larger cohorts.”
Alex Aravanis, Illumina’s chief technology officer and head of R&D, next took to the stage to talk about the multiple breakthroughs that made the NovaSeq X series a reality. “Our goal was to make a platform that powers you to make the next scientific breakthroughs, to discover new dimensions of biology, such as epigenetics and proteomics, and to accelerate our understanding of the causes of disease and find new ways to treat them and cure them, and we knew the existing platform couldn’t get you there,” he says.
“Over five years, we reinvented everything, every single piece of technology, from chemistry to engineering,” Aravanis says. “It started with the sequencing chemistry to achieve a massive increase in output for NovaSeq X. We needed smaller, denser DNA clusters to be blasted with high-power lasers, [and] cold chain logistics ... had to go.”
While Illumina’s SBS chemistry is the industry standard, when tested under the required extreme conditions, it “just wasn’t stable enough,” he says. So, the team that originally invented SBS was challenged to come up with a new SBS that could be driven harder and faster to create thousands of chemical structures.
“We also needed a new DNA polymerase that could rapidly incorporate the new nucleotides,” he quickly adds. The team in fact created millions of candidate polymerases to find the perfect one. Illumina spent “more time, money, and scientific power in the development of XLEAP than had ever been done before in history.”
This discovery, together with fully functional nucleotides that are twice as fast, enable XLEAP to do single-day runs, says Aravanis. The chemical bonds of XLEAP are 50 times more stable and thus able to withstand more intense lasers. That translates into higher accuracy, the potential for longer runs, and the ability to ship at ambient temperatures.
XLEAP-SBS is the first-ever SBS chemistry that can be shipped over a wide range of temperatures—from minus 20 degrees Celsius up to 50 degrees—and the cartridges are made of more recyclable plastics, he says. In addition to being biodegradable, the cartridges are easy to use with single addressable lanes so researchers can do many combinations of experiments.
Ultra-Dense Flow Cells
Illumina still needed to build flow cells that could pack more sequencing in smaller areas, to give end users more genomes in a single run, continues Aravanis. To get to 25 billion single sequencing reads, the density had to be 320% bigger than the existing NovaSeq. No technology or manufacturing processes existed that could make that type of ultra-dense flow cells, so Illumina’s flow cell team developed one with tens of billions of nanowells at fixed locations.
The semiconductor industry would help build the nano-scale components, allowing Illumina to move to a bigger, 300-millimeter wafer and automate the manufacturing process, says Aravanis, “pushing throughput scale and reducing variability effects.” Coupled with the increased flow cell yield, that lowers cost of use.
To ensure adequate supply and “exquisite” process control, Illumina built state-of-the-art manufacturing facilities in the U.K., Singapore, and San Diego, he continues. This will help lower the cost of XLEAP consumables and flow cells that will be used on NovaSeq X as well as Illumina’s forthcoming NextSeq instruments.
Accuracy Plus Speed
An entirely new imaging system also had to be created to take full advantage of the new-and-improved chemistry and flow cells, says Aravanis. The system has higher sensitivity and resolution, four times more pixels and twice the data rate, and can scan the length of a flow cell in one second while maintaining nanometer-scale focus, which he likens to “throwing a baseball from here in San Diego to London and only being off by the thickness of a human hair.”
Optical engineers at Illumina invented high numeric aperture optics—"the highest resolution ever for a SBS sequencer”—for NovaSeq X, Aravanis shares. That, in turn, required a new focus tracking system with sub-50 nanometer accuracy.
NovaSeq X can image a lane from top to bottom in under a second and do so thousands of times per run, says Aravanis. “That means we are scanning 10 times more DNA in clusters per second than you can do right now.”
To keep pace with this sequencing speed, the Illumina team had to double the rate at which imaging data was converted into base calls, from 44 hours using NovaSeq 6000 to less than 24 hours on a NovaSeq X, Aravanis says. Additionally, they upgraded computing components (including the latest multicore processors) to give users the flexibility to run multiple flow cells in overlapping runs.
A lot of work goes into extracting insights from sequencing data, so merely giving users more data would have only compounded the problem, Aravanis continues. “So, we revolutionized what happens after sequencing but still on the sequencer” by integrating the DRAGEN platform to process up to 128 genomes in a single run. “Every NoveSeq X now shares a supercomputer on board.”
The Illumina team did all this without making NovaSeq X any bigger or heavier or more power hungry, he quickly adds. There was no space to spare to start with, so engineers miniaturized every component to pack it all in. “Now, for the first time ever, before the run is even finished the system converts the native base calls into the industry-standard FASTQ format.”
The NovaSeq X machine also slashes the amount of processing heretofore required after the run ends, says Aravanis. “This means fewer staff to manage the data, and you pay a lot less to get your answer. Within hours of sequencing completion, you get the most accurate and comprehensive genome all produced on NovaSeq X without having to do anything more.” Importantly, all the reengineering translates into a fivefold reduction in users’ data footprint and storage costs.
On rare cases where hard-to-reach regions are being sequenced, there’s no need to toggle to a different machine, Aravanis points out. Illumina Complete Long-Read technology offers improved accuracy even in FDA-defined benchmark regions.
Since contiguous long reads aren’t needed for many applications, he adds, Illumina scientists are now developing a kit allowing users to target only their regions of interest. “You won’t have to sequence what you don’t need, and you’ll be able to start using this kit by the end of next year.”
In his wrap-up, deSouza notes that early adopters of NovaSeq X cite its promise in sequencing larger, more diverse cohorts and even the genomes of entire nations to help unravel the genetic basis of common diseases. It is viewed as potentially transformative for clinical and research operations and could accelerate the path to a $100 genome. Regeneron, a longtime partner of Illumina, expects the NovaSeq X will help it sequence tens of millions of exomes and genomes soon, he adds.
“When you look at the NovaSeq X, you see our most sustainable, most powerful sequencer ever, and I also hope you see the dream and breakthroughs and lives you will change with this platform,” he tells his audience. “The true power and potential of NovaSeq X is only possible with partnership ... [and], together, we are going to change the world.”