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Agencourt, USDA, JGI Comparing ABI, 454, Illumina Tools for Microbial Re-Sequencing

Agencourt Bioscience is working with scientists at the US Department of Agriculture and at the Department of Energy’s Joint Genome Institute to test and compare Applied Biosystems’, Illumina’s, and Roche/454’s next-generation sequencing platforms for whole-genome microbial re-sequencing, In Sequence has learned.
Last month, the Beckman Coulter subsidiary presented preliminary data of the project, generated on the ABI SOLiD platform, at the Advances in Genome Biology & Technology conference in Marco Island, Fla.
Separately, Agencourt, which claims to be the largest provider of contract sequencing services, said it plans to bring more next-gen sequencing platforms in-house, Doug Smith, Agencourt’s director of grant development, told In Sequence last week.
In their ongoing project, Agencourt and its collaborators plan to resequence a mutant form of the yeast Pichia stipitis on the SOLiD platform, 454’s Genome Sequencer, and Illumina’s Genetic Analyzer, and want to compare the results with the wildtype genome.
P. stipitis, which has a 15.4-megabasepair genome spread over eight chromosomes, is good at breaking down xylose, a major component of wood, and could have applications in bioconverting wood into ethanol.
 “We are using this as a project to evaluate all the available next-generation sequencing technologies because it’s a good-sized genome and we have a finished sequence for it,” said Smith.
The data from the SOLiD platform he presented last month were some of the first generated for scientists outside of ABI and presented in public. Other independent researchers showing data from the SOLiD platform at the meeting were Ian Goodhead and Chris Clee from the Wellcome Trust Sanger Institute (see In Sequence 02/20/2007),  and Arend Sidow from Stanford University Medical School.
In a paper that published online in Nature Biotechnology last weekend, the Agencourt collaborators, led by Thomas Jeffries at the USDA’s Forest Products Laboratory and Paul Richardson at JGI, described the genome sequence of wildtype P. stipitis. They generated the sequence by capillary electrophoresis sequencing, using a shotgun approach followed by high-quality finishing.
The Agencourt researchers used ABI’s SOLiD platform to sequence a mutant strain of the yeast that can ferment xylose better than the wildtype. According to Smith, Jeffries’ laboratory generated this mutant several years ago in a three-stage protocol that involved a targeted disruption of a particular gene and two rounds of random chemical mutagenesis followed by selection.
ABI’s high-throughput genomics group in Beverly, Mass. — the former Agencourt Personal Genomics group that shares the same building with Agencourt Bioscience — performed the sequencing using a prototype of the instrument ABI plans to commercialize later this year. They collaborated with the Agencourt researchers to analyze the data.
The researchers generated more than 600 megabases of sequence in a single run, constituting 30x coverage, and aligned the 25-base-pair reads back to the finished wildtype sequence using ABI’s color-space alignment algorithm, “which is a unique feature of the platform,” according to Smith.

P. stipitis is able to break down xylose, a major component of wood, and could have applications in bioconverting wood into ethanol.

“The coverage was quite uniform across the whole genome, except in regions of repetitive sequence,” he said.
The researchers also detected approximately 25 SNPs, which, “based on a number of parameters, were high-confidence candidate mutations,” he said.
In order to confirm these mutations, the Agencourt researchers re-sequenced the mutant with their in-house 454 Genome Sequencer 20 and were able to confirm “all of the ones that we had data for,” Smith said.
Because they only sequenced the genome to 3x coverage with the 454 platform, they were not able to say with confidence whether the ABI platform missed any SNPs that 454’s was able to pick up, but “we will probably do that analysis in the future,” he said.
The researchers are now using the SOLiD platform to generate paired-end data, are creating more 454 data, and resequencing the mutant on their Sanger platform.
The JGI collaborators are generating data on the Illumina platform, Smith said. In addition, the scientists would like to go back to the intermediate strains of the P. stipitis mutagenesis and “see the history of mutations as they were generated through that process.” They hope to finish the project by the end of spring.
The company is also working on “a couple” of other projects involving the SOLiD platform, Smith said, but could not provide more details yet.
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