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Illumina Targets HLA Typing, Forensics with MiSeq; Unveils NeoPrep, Details about New Sequencers


Illumina announced a new automated sequencing library prep system called NeoPrep last week, along with plans to sell customized versions of the MiSeq system for forensics and HLA typing.

During a meeting with investors, which was webcast, the company also revealed further details about the NextSeq 500 and HiSeq X Ten sequencing platforms it announced earlier in the week.

In addition, the firm provided an update on its strategy for expansion into new markets, especially in the clinical arena.


The NeoPrep library prep system incorporates electrowetting technology from Advanced Liquid Logic, which Illumina acquired last summer.

The new benchtop system, which is in the final stages of development, will launch this summer and has a target list price of less than $50,000, according to Kirk Malloy, general manager of Illumina's life sciences business unit, who introduced the product.

It will initially be available for the three most popular library preparation protocols, to be expanded to six protocols by the end of this year, he said. The system can take RNA or DNA, up to 16 samples per run at launch, and can work with as little as 1 nanogram of input material for some assays.

NeoPrep libraries will be compatible with all Illumina sequencers and will be "fully integrated" with the BaseSpace cloud-based informatics platform.

The instrument uses electrowetting digital microfluidics developed by ALL. All reactions take place on cartridges that require no pumps or tubing. "The opportunities for human error are virtually eliminated," Malloy said.

NeoPrep reduces hands-on time, number of steps, and reaction volumes required for library preparation, and increases the reproducibility of protocols, he said.

In terms of sample prep reagents, Illumina plans to launch a kit this year that will enable synthetic long reads, based on the Moleculo technology, and a TruSeq-based kit that addresses the market for researchers using formalin-fixed, paraffin-embedded tissue samples, in particular oncology customers, he said.

HLA Typing, MiSeq FGx

Illumina also plans to enter two new emerging markets with dedicated MiSeq-based systems for HLA typing for transplant medicine and forensics.

In mid-2014 it plans to launch a TruSight HLA system consisting of a sample prep kit for eight HLA genes, which will be fully-phased; the MiSeq or MiSeqDx instrument; and integrated analysis software from an unnamed "market-leading" provider.

According to Matt Posard, general manager of Illumina's new and emerging business unit, the HLA-typing market is about $250 million in total and "ready for a new technology that can address the shortcomings of the existing technology."

Within the first half of the year, Illumina plans to launch the MiSeq Forensic Genomics system, or MiSeq FGx, consisting of a forensic PCR and library preparation kit, a version of the MiSeq with forensic-specific applications and a unique user interface, and software for primary and secondary data analysis as well as semi-automated data interpretation.

The software was designed with input from forensics experts from regional and national crime laboratories. Of note, it will be backward-compatible with the Combined DNA Index System, CODIS, the Federal Bureau of Investigation's criminal DNA database program, as well as other criminal databases, Posard said, which is crucial to forensics customers.

Like existing capillary electrophoresis-based technology, the MiSeq FGx will interrogate short tandem repeats, or STRs, but also evaluate other genetic markers from 200 loci that provide ancestry information and physical traits.

"Like the HLA market, we think this [market] is mature but it's also grossly underserved," Posard said, noting that the total market is about $400 million. Current technologies often cannot differentiate material from several individuals in the same sample, he said, or obtain results from partially degraded samples.

The MiSeq FGx will be able to work with sub-nanogram amounts of input DNA, distinguish DNA from different individuals, and provide additional information besides established STRs.

Over the next five years or so, Illumina aims to convert about half the forensics market to next-gen sequencing, Illumina CEO Jay Flatley said during the investor event.

HiSeq X Ten

As announced last week, the HiSeq X Ten, which will be sold in sets of 10 for $10 million, can generate up to 1.8 terabases of data per system in under three days and will be licensed to customers for whole-human genome sequencing only. Reagent costs are about $7 per gigabase, compared to $43 on the HiSeq 2500 at present.

It is Illumina's first sequencer that uses ordered arrays, a technology the company announced a year ago and tested with early-access customers at the end of 2013, according to Flatley.

The company plans to sell a total of five HiSeq X Ten systems this year due to supply constraints for the high-performance cameras and ordered arrays; however, Flatley said during the event that "if we're more successful in ramping that supply chain, we may have the ability to deliver somewhat more of these into the field" this year.

Customers sign a legal agreement with Illumina that only allows them to use the instrument for human whole-genome sequencing. The company can keep an eye on customers' use of the machines because their number will be small, and because Illumina service engineers will be present at these customer sites "on an absolutely routine basis," sometimes even with their own offices, Flatley said, "so we will be seeing day to day what they do with these products."

In addition, there is no automated workflow for preparing exomes or other types of samples in large enough numbers for the HiSeq X Ten right now, he said. Over the next year and a half or so, Illumina plans to work on such protocols with collaborators, he said, and will "very likely" open up the HiSeq X Ten to other applications in the future.

Finally, the patterned flow cells and HiSeq X Ten sequencing chemistry have so far only been tested for whole-human sequencing and would need to be optimized "to some extent" for other applications, he said.

Since the HiSeq X Ten was announced last week, the company has been "getting tremendous interest from the field in this product," he said.

The system generates about 120 gigabases of data per flow cell lane, equivalent to a human genome at 30X coverage. Per two-flow cell run, Illumina scientists generate about 1.9 terabases of data with "consistent coverage per lane," Flatley said, but the company quotes a conservative spec of 1.8 terabases.

Flow cells with ordered arrays are now routinely being manufactured at a commercial foundry, he said, where 12 flow cells are cut from one wafer. With ordered arrays, the company has control over the size of the clusters, their pitch, or distance between them, and their position.

Each array consists of nanowells that are 400 nanometers in diameter and spaced about 700 nanometers apart, center to center. This increases the density of clusters by about twofold compared to random arrays, from 770,000 per mm2 to about 1.6 million per mm2. The features are present both on top and at the bottom of the flow cells.

Going forward, Illumina will be able to reduce the size of the features as well as their pitch. "We picked this initial 400/700 combination because it's working very well, it's in a safe zone to allow us to scale up the technology initially," Flatley said.

The system will also use a new cluster generation chemistry. Flatley explained that the number of nanowells, or pads, that are occupied by a single DNA template during the seeding stage — which is needed for sequencing — is limited to about 40 percent by Poisson statistics.

"The way we overcome this is through simultaneous seeding and amplification," he said. As soon as a template molecule lands on one of the nanowells during the seeding phase, it is "virtually instantly amplified," he explained, using an amplification process that is about an order of magnitude faster than the seeding process.

If a second template tries to get onto a nanowell that is already occupied, it gets rejected. As a result, 75 to 80 percent of the nanowells are monoclonal. "This was a key breakthrough that we have been working on for a couple of years," Flatley said.

The system's sequencing chemistry will also be "much faster" than before, due to an improved DNA polymerase with "great accuracy and great speed."

To bring the cycle times down, the company took out "dead time," for example by speeding up the paired-end turn when using paired-end kits.

The system's camera, which scans six times faster than the camera of the HiSeq 2500, is customized and was co-developed "over many years" in partnership with "an outside supplier," Flatley said, noting that the ability to do bi-directional scanning is a "key breakthrough" in this camera.

The HiSeq X Ten also has higher-power lasers than other Illumina sequencers to increase the signals, allowing for faster scanning.

The system's computers are faster, too, to be able to handle the fast data generation, and Illumina created new software to analyze the new types of images coming from the patterned flow cells.

NextSeq 500

As previously reported, Illumina's other new sequencing system, the NextSeq 500, has a list price of $250,000 and features a new sequencing chemistry with only two instead of four colors - red and green. The first unit was shipped to a customer last week.

The instrument can generate up to 40 gigabases of data in 26 hours with mid-output flow cells or up to 120 gigabases of data in 29 hours with high-output flow cells. The reagent cost per gigabase is about $33.

It targets applications like exome sequencing, counting applications for NIPT, and gene panels, although amplicon sequencing and human genome sequencing are also possible.

Christian Henry, Illumina's chief commercial officer, said that the NextSeq was developed over the last two years from scratch, with over 300 people contributing. It is about the same size as the MiSeq, uses reagent cartridges, and is "deeply integrated" with the BaseSpace informatics platform.

Unlike other sequencers, which were initially manufactured in the US, the NextSeq is built at Illumina's Singapore factory from the start, though the flow cells and reagents are manufactured in San Diego, at least for now, partly because custom-built robotics are required to fill the reagent cartridges.

The system's flow cells — each with four lanes, which differ in width between the mid-output and the high-output flow cell — are larger than those of existing Illumina sequencers and are encased in a plastic cartridge to protect them and make them easier to handle. They use color-coded icons to help users match them with the correct reagent kit and can only be inserted in one orientation.

The flow cells have a new surface chemistry, allowing them to be shipped dry, "which was a major, major request of our customers," Henry said. This also makes storage and shipping easier and more convenient for Illumina, Flatley added.

The instrument only takes two images instead of four per cycle, resulting in faster sequencing. Illumina compared RNA sequencing data from the same libraries sequenced on the NextSeq and the MiSeq and found the datasets to be "practically identical," Henry said. Data from NextSeq and HiSeq were also very similar, he added.

Six optical modules, which contain LED light sources and custom-designed optics, are combined into one optical unit. The units are manufactured by an outsourced factory and are about a third of the cost of a HiSeq camera, light-weight, and require no alignment on arrival at a customer's site, unlike the cameras of MiSeq and HiSeq instruments, Henry said.

The fluidics module requires no tubes, is easily swapped out, operates with lower volumes, and has lower maintenance than the fluidics systems of Illumina's other sequencers. The company built a new fluidics technology that allows it to perform chemistry and imaging simultaneously on the same flow cell, resulting in reduced run times. At the end of each run, the fluidics system undergoes an automated wash cycle.

Expansion Plans

This year, Illumina plans to expand into new markets, especially on the clinical side, and sees opportunities in markets that total $20 billion this year, according to Henry.

Oncology is about a $12 billion market, including $8.5 million from theranostics, which consists of clinical diagnostics and companion diagnostics; about $2 billion from basic and translational cancer research; about $1 billion from inherited cancer analysis; and about $500 million from molecular monitoring, such as the detection of residual disease.

The life sciences market is about a $5 billion opportunity, Illumina said. According to Malloy, about $1.5 billion of that derives from the core research market and genomics labs, and more than $2 billion from complex disease research, which includes rare variant association studies as well as integrated analyses of the genome, transcriptome, and epigenome; immune repertoire screening; and single-cell analysis. The agrigenomics market represents an opportunity of about $500 million, including research and screening in livestock and crops. Another $500 million comes from microbiology and metagenomics, he said, in particular complex microbiomes like the human gut.

Reproductive and genetic health is about a $2 billion opportunity, Henry said, with about $1.1 billion from NIPT, $700 million from IVF —both preimplantation screening and preimplantation diagnostics — and $200 million from neonatal and carrier screening.

New and emerging markets represent about $1 billion in opportunities, with about $300 million from HLA testing, $400 million from forensics, $100 million from consumer markets, and the remainder from unidentified markets.

According to Henry, Illumina increased its direct sales force by 27 percent in 2013, and its field staff by 67 percent. This year, the company plans to grow its field force further, "roughly at the rate of our revenue growth," Henry said. It is also combining its general and clinical sales forces, which were separate for the last several years.

Last fall, Illumina revealed a plan to restructure the company into five business units — Life Sciences, Reproductive and Genetic Health, Oncology, Enterprise Informatics, and New and Emerging Markets — in part to address new markets more effectively. As part of the reorganization, it also centralized product development, IVD product development, operations, and quality control across the company. All of those changes took effect Jan. 1.