At AGBT, Illumina Provides Additional Details on Project Firefly

ORLANDO (GenomeWeb) – At this week's Advances in Genome Biology and Technology meeting in Orlando, Florida, Illumina CEO Jay Flatley provided additional details about Project Firefly, the company's semiconductor sequencing platform unveiled in January, which Flatley said today could eventually be integrated in a hospital setting.

Firefly will "open the next level of the market because it's so simple," he said in a presentation. The ultimate goal is to have a device where the input is a raw sample and the output is a report. Although Illumina is "not there yet," Firefly is "certainly one step in that direction," and the firm is working on the elements that would get to that spot, said Flatley.

In response to an audience member's question about a handheld sequencing device, Flatley said that it is "on our roadmap."

As the company previously said, Firefly will be based on CMOS technology the firm acquired when it bought Avantome in 2008.

Illumina got far in the development of Avantome's technology, Flatley said, but never commercialized it because they were unable to get it to work without the use of emulsion PCR. However, Illumina continued to work developing a sequencing-by-synthesis chemistry that could work on a semiconductor chip.

The Firefly device is essentially a CMOS sensor with nanowells. The nanowells are fabricated over photodiodes to enable DNA deposition to be aligned one-to-one with each photodiode. Clustering and sequencing then occurs directly on the CMOS chip.

Because CMOS is "inherently a one-channel device", Flatley said the firm's researchers had to figure out how to develop a one-channel sequencing-by-synthesis chemistry.

Firefly's chemistry will use a new encoding technology. In its four-channel chemistry, which is used by Illumina's HiSeq family of instruments, each nucleotide is labeled with a separate fluorescent dye that are detected in four different optical channels. When Illumina launched NextSeq, it introduced a two-channel chemistry. That chemistry used two fluorescent dyes in such a way that guanine was always dark, adenine and cytosine each had a single dye, and thymine had two dyes.

In the one-channel chemistry, thymine will have a permanent fluorescent label. Adenine will have the same fluorescent label, but that dye will be removable. Guanine will be permanently dark. And, the cytosine will start out dark but will be tagged so that a dye can be added to it.

Flatley then demonstrated how this scheme would work to read the DNA. Essentially, in a first image of the four nucleotides, A and T are both labeled and detectable. Then, in the second image, the dye is cleaved from A and added to C. In the second image, only C and T fluoresce. By combining the information from the two images, all four bases are easily discriminated, Flatley explained.

In internal tests, Flatley said that the firm has already demonstrated 99 percent raw read accuracy with paired-end 2x150 base reads, comparable to the performance on the HiSeq X.

Although the company does not plan to launch Firefly until the second half of 2017, Flatley said the company decided to disclose its plans early because it will be ramping up its external supply chain, which would be difficult to keep a secret. In addition, he said that the company will be working with third-party developers starting in the second half of 2016 and expanding the collaborations in 2017 in order to expand the number of applications that can be run on the platform.

The platform itself will consist of two modules totaling one cubic foot of volume. One module will be for library prep and will be able to prepare up to eight libraries in parallel in 3.5 hours, unattended. The library prep cartridge will leverage digital fluidics technologies that are being used in Illumina's NeoPrep, an instrument launched last year at AGBT that performs library prep, quantification, and normalization.

The reagents will all be pre-sorted into pods and automatically delivered. The user will just have to load the sample and the primers. The device will be able to deliver eight separate libraries or can multiplex the samples into one library for sequencing. In addition, one library prep module can support up to eight sequencing modules.

The libraries are then loaded into the sequencing cartridge, which contains the CMOS chip. Sequencing will take between 3.5 hours and 13 hours, depending on the application, and then the results will be loaded to Base Space, Illumina's computing environment for next-generation sequencing data analysis and management. The system will be driven by an iPad so that it can be monitored wirelessly, Flatley said.

Firefly's 1 gigabase of output will make it "ideal" for applications that include "targeted discovery, resistance monitoring, and PGS," Flatley said.

The cost will be less than $30,000 for both modules and around $100 per sample.

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