Sequencing by Synthesis
Like other awardees, Agencourt is taking a sequencing-by-synthesis approach, in which measurement takes place as nucleotides are incorporated into DNA. The firm's technology is based on polony sequencing, short for polymerase-generated colony, originally developed in the laboratory of George Church, Ph.D., at Harvard University.
Agencourt has optimized methods for bead-based polony sequencing. In this system, clonal populations of short DNA fragments are amplified onto beads, then packed tightly onto a slide containing a gel matrix. In a flow cell, reactants flow over the slide to allow DNA synthesis to take place.
"Hundreds of millions of DNA beads can be sequenced in each run," says Dr. Costa. "Our technology speeds up conventional sequencing by 100-fold, and will be the equivalent of 500 to 1,000 ABI 3730xl instruments a day."
Agencourt's methodology, like other non-single molecule sequencing by synthesis approaches, affords read lengths of only 50 to 150 base reads. This makes sequence assembly more challenging. To get around this, Dr. Costa says the company uses "tricks" to get paired end information across short reads, which allows researchers to put short segments of nucleotides "together like a jigsaw puzzle."
"What really sets us apart is our simple and robust biology," Dr. Costa says. "Our system is not complicated. Simplicity and consistency are important attributes."
454 Life Science is "refining and advancing the performance of sequencing by synthesis," according to Marcel Margulies, vp engineering. Margulies says 454's technology is extremely high throughput, both in terms of sequencing reactions and front-end sample preparation.
"We perform sequencing by synthesis on solid support and on 400,000, 500,000, and even 700,000 clonally amplified fragments simultaneously," he says. "Entire floors of robots" can be devoted to sample preparation for conventional sequencing.
"With our approach, irrespective of the size of the genome, you can do library preparation with one person, with a single sample preparation, in a couple hours," says Margulies.
DNA is amplified and bound to beads, which are then deposited into a fiberoptic plate with 1.6 million wells. Because of size exclusion and a 2.5 times oversupply of wells, only one bead gets deposited into each well. Nucleotides are then added in sequence. When incorporated, inorganic pyrophosphate is released.
"That release is observed through emission of light that is captured. Measurement takes place at each incorporation. The beauty is that if you have a homopolymeric stretch, all will incorporate at the same time, and you get five times as much light," says Margulies.
Margulies says 454 generates a tremendous amount of data because of the number of reads performed simultaneously. To address this, they have outfitted computers with FPGA (field programmable gate array) chips.
"The entire computer takes advantage of this to do data processing in real time. The way we designed the system, at the end of the run, we have reads that have been corrected and quality scored."
454 has also developed its own assembly software. "Typically people convert signal to nucleotide letters, and then use the letters to do all the manipulations. We generate that as well, so one could use that information in conventional assemblers or mappers.
"But this may not ultimately be the best way of doing things. We have found that if you use signals themselves to find homologies as opposed to the letters, you can do a better job," Margulies explains.