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Abbott Subsidiary Ibis Biosciences Developing Microfluidic Sample Prep System for Next-Gen Sequencing

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By Ben Butkus

BALTIMORE, Md. – Abbott subsidiary Ibis Biosciences is developing an integrated microfluidic sample prep card and instrument that lyses cells, extracts nucleic acids, and performs whole-genome amplification to generate starting material from trace specimens for whole-genome sequence analysis, a company representative said at a sample prep conference here this week.

The technology is currently in beta-testing, but researchers at Ibis have already demonstrated that the system can generate sufficient templates for analysis on a number of next-generation sequencing platforms, including those sold by Roche 454 and Pacific Biosciences.

Mark Eshoo, director of new technology development at Ibis, shared details of the company's technology during a presentation at the Knowledge Foundation's Sample Prep 2010 conference, held here this week.

Eshoo did not provide a commercialization timeline for the sample prep system; however, it would be the first major new product that the company has disclosed since Abbott acquired it in January, primarily for Ibis' T5000 Biosensor System, which is used for rapid identification and characterization of infectious agents.

That system has been reborn as the Abbott Plex-ID system, for which Eshoo leads bioprocess development.

Eshoo said that Ibis began developing the sample-prep system to address existing challenges of next-generation sequencing, which include the fact that up-front sample prep can take several days; nucleic acid contamination can be "fatal" to the sequencing project; and many specimens may consist of only a few cells, such as when researchers are analyzing tumor samples or attempting to sequence DNA from viruses or bacteria, Eshoo said.

For instance, to perform a whole-genome sequencing reaction on DNA obtained from various sources, "using Roche 454 technology, you'd need 1 million human cells' worth of DNA; 108 [Bacillus anthracis] cells; or 1010 smallpox viruses, which is actually one of the largest virus genomes," said Eshoo.

Sample prep for Roche 454 sequencing, he said, "is quite laborious and takes many days. Under the best case scenario, it's a three-day process; but for most real samples, it can take five to seven days to prepare a sample just to go into the sequencer."

And although the sample prep front end for Pacific Biosciences' sequencing technology is "much simpler," Eshoo said, it still requires a first day of sample prep that generally entails sample pre-treatment, cell lysis, and nucleic acid extraction.

"This is highly technical, takes a lot of equipment, and a lot of lab space. It's not a fun process," he said.

Ibis is currently developing its microfluidic card and accompanying instrument to "integrate this one-day process, and reduce that down to less than an hour," Eshoo said. "We're looking at the cell lysis, the nucleic acid extraction, and whole-genome amplification integrated into the microfluidic card. Ultimately, we want to take that entire process and integrate it into a microfluidic system."

Eshoo said that the microfluidic card systems comprise different laminated "sub-circuit" cards for cell lysis, DNA extraction, and whole-genome amplification, with each sub-circuit easily "changed on the fly" to fit specific applications. These circuits are then plugged together to form an integrated circuit that performs all prep steps in a continuous single process.

Integrating microfluidic transport offers up several advantages over current sample prep methods, he said. For instance the cell lysis and DNA extraction sub-circuit uses "standard chemistry like you would do using [a] Qiagen [kit], but we are able to get very good efficiency from this kind of a format," Eshoo said. "Because of the microfluidics, you can do things you can't do with a single pass-through column. You can pass the reagents over a membrane multiple times to get maximum binding efficiency; and conversely you can get maximum elution efficiency, too."

Meantime, the whole-genome amplification sub-circuit, which uses a method called multiple displacement amplification, features ultra-clean reagents and improved buffer formulations, random primers, and enzyme formulations.

The various reagents are contained in foil "blister packs" on the outside of the card, and during the process are released into the appropriate microfluidic channels and chambers to perform various reactions. The whole card plugs into the side of an instrument — currently in beta-testing — that is about the size of a standard benchtop themal cycler and drives the various pneumatics and pumps for all of the microfluidic reactions.

So far, Ibis has tested the lysis and DNA isolation capabilities of the system using anthrax spores, Staphylococcus aureus, and Klebsiella pneumonia samples, and was able to extract plentiful DNA from the organisms in about 5-minute reactions.

Meantime, "we wanted to demonstrate that our whole-genome amplification of DNA did not introduce errors" for downstream sequencing analysis, so Ibis tested the system for use with three different sequencing platforms: Sanger sequencing, Roche 454, and Pac Bio.

In each case, their results varied little from reference DNA databases, and sequence analysis following the integrated sample prep procedure yielded similar results in terms of SNPs and genome coverage.

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