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Ion Torrent Team Publishes Semiconductor Sequencing Study

By a GenomeWeb staff reporter

NEW YORK (GenomeWeb News) – Researchers from Life Technologies' Ion Torrent published a paper in Nature online today outlining how they used their semiconductor sequencing strategy to sequence bacterial genomes and an individual human genome — that of Intel co-founder and "Moore's Law" author Gordon Moore.

The team used the Ion Torrent sequencing system, which uses an integrated circuit semiconductor system developed using the "complementary metal-oxide semiconductor," or CMOS method, to sequence the genomes of three bacterial species — Vibrio fisheri, Escherichia coli, and Rhodopseuomanas palustris.

They scaled up their method to sequence Moore's genome by developing ion chips that contained up to 10 times as many sensors as those used to sequence most of the bacterial genomes, which they then analyzed and compared to a genome sequence for the same individual that had been generated on the ABI SOLiD platform.

Ion Torrent Founder, CEO, and Chairman Jonathan Rothberg, first author on the study, presented preliminary findings from the Moore genome project at the Molecular Medicine Tri-Conference earlier this year. The new paper marks the first publication of this work.

"We have demonstrated the ability to produce and use a disposable integrated circuit fabricated in standard [complementary metal-oxide semiconductor] foundries to perform, for the first time, 'post-light' genome sequencing of bacterial and human genomes," Rothberg and co-authors wrote.

Rather than relying on optical or imaging technology, the Ion Torrent platform uses chips containing millions of wells and sensors, the authors explained.

"The all-electronic detection system used by the ion chip simplifies and greatly reduces the cost of the sequencing instrument," they wrote. "The instrument has no optical components, and is comprised primarily of an electronic reader board to interface with the chip, a microprocessor for signal processing, and a fluidics system to control the flow of reagents over the chip."

Genomic DNA that's been fragmented and linked to adapters is amplified and linked to beads. Once these "template-bearing beads" are enriched, researchers toss in polymerase enzyme and sequencing primers and load the lot into a sequencing chip. Spinning the chip in a centrifuge, nudges these beads into the chip's sensor wells.

As each nucleotide base is added sequentially, the nucleotides that are complementary to template DNA get incorporated into a growing DNA strand that's been initiated by the sequencing primer. The polymerase releases hydrogen ions as these nucleotides are added, changing the pH of the solution. These protons are then detected by metal oxide sensors linked transistors, leading to a shift in voltage that's ultimately converted to base calls by off-chip electronics.

"Sequence data are obtained by directly sensing the ions produced by template-directed DNA polymerase synthesis using all-natural nucleotides on this massively parallel semiconductor-sensing device or ion chip," the study authors explained.

"The ion chip contains ion-sensitive, field-effect transistor-based sensors in perfect register with 1.2 million wells," they added, "which provide confinement and allow parallel, simultaneous detection of independent sequencing reactions."

To take a crack at de novo genome sequencing using this method, the researchers first sequenced the genomes of a V. fisheri strain to 6.2 times coverage and a R. paustris strain to 6.9 times coverage. Using larger and larger ion chips, they also sequenced the E. coli genome three times to between 11.3 and 58.4 times coverage.

The per base accuracy of the genomes was more than 99.6 percent for the first 50 bases of each read and roughly 98.9 percent for the first 100 bases, researchers reported. The accuracy dipped slightly in parts of the genome containing homopolymer runs, but was still around 97.3 percent accurate across a five base homopolymer. Read lengths were typically around 100 bases, though some were as long as 200 bases or more.

The team then scaled up the number of sensors in the chips to tackle a human genome, sequencing Moore's nuclear genome to a mean of 10.6 fold coverage using roughly 1,000 ion chips. In the process, they also got 732 times coverage of his mitochondrial genome.

During their analyses of the nuclear genome, the team found nearly 2.6 million SNPs. Of these, 99.95 percent of the heterozygous and 99.97 percent of the homozygous genotypes were verified by ABI SOLiD-generated sequence that covered the genome about 15 times.

The genome also contained 3,413 of the 7,693 inversions and deletions detected by members of the 1000 Genomes Project, as well as certain SNPs found in either the Online Mendelian Inheritance in Man database or a 23andMe database of functionally important SNPs.

"By demonstrating the ability to make larger and denser arrays, use fewer transistors per sensor, and sequence from wells as small as 1.3 [micrometers], our work suggests that readily available [complementary metal-oxide semiconductor] nodes should enable the production of one-billion-sensor ion chips and low-cost routing human genome sequencing," the researchers concluded.

Sequence reads from the Moore genome have been deposited into the Sequence Read Archive.

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