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BIODEFENSE BRIEFING: Perlegen Tiles Through Salmonella Sequence; 454 (Quietly) Touts Biodefense Applications of Its Microbial Sequencing Technology

This article has been updated from a previous version.


NEW YORK, Sept. 25 (GenomeWeb News) - Under a recent biodefense grant from NIAID, Perlegen Sciences is planning to tile through the genomes of 40 different strains of Salmonella with its microarray wafers, in order to obtain signatures of SNP sets, a company official said this week.


"We are looking at different [Salmonella] pathogens, where there is the issue of 'how do you quickly identify where the strain came from?', so you can have attribution and know the source of an outbreak," said Paul Cusenza, vice president of alliance management at Perlegen.  The point of the research  "is to understand the genetic fingerprint of each strain," he said, "so we can understand what strain is present."


The grant, according to the NIH website, is for $500,000.


Perlegen, a Mountain View Calif., genotyping company that spun off of Affymetrix in 2000,  is working on this project with Gary Andersen of Lawrence Berkeley National Labs, an expert on Salmonella and other pathogens who has previously developed PCR-based tests for rapid identification of Salmonella. The team will select 40 different isolates within three different serovars, or subtypes, based on existing knowledge about the diversity of the strains, Andersen said. They will then resequence these on super-sized Affymetrix arrays that contain oligo probes representing, collectively, the full sequence of four fully sequenced strains of Salmonella.


The wafer arrays, which contain 60 million probes and are each 5' x 5', are so big "they blow my mind away, said Andersen. "60 million probes is just phenomenal. It's not even micro any more."


This reference sequence on the arrays comes from a strain that Andersen's lab has sequenced, as well as from three strains sequenced by other groups around the world. (Researchers at the Sanger Center, Taiwan's Chang Gung University, Washington University , the University of Illinois and University of Wisconsin are all involved in Salmonella sequencing projects.)


The wafer technology, said Andersen, is "perfect" for a project to identify genetic variants of Salmonella, said Andersen, "because the differences between the different isolates of salmonella at the gene level are very minor, [and] there are very few differences. You can go along for 10kb without any difference in the genome sequence at all." In addition to enabling researchers to detect SNPs, it also allows them to detect deletions relative to the strains that are on the chips, as well as insertions that happen to also be on one of the strains on the chips (although it does not allow detection of insertions not on any of the four reference strains.)


Andersen's collaboration on the project emerged from his professional relationship with David Cox, Perlegen's co-founder and chief scientific officer.  "We've had mutual interests for over a year now, " Andersen said. "I also work on hybridization arrays in my lab at Lawrence Berkeley Lab ... I've looked at it from a more microbial end, whereas he's done it from the human end. It seemed like a good mesh."


This project follows on Perlegen's project to resequence and compare up to 50 haploid copies of the human genome on the wafers of arrays, said Cusenza. In the human genome resequencing project, company scientists are comparing different genomes to find out what genetic variants are present, and doing SNP association studies for disease and drug response. When this project was progressing, the company realized "the same technology we used could also be used to [resequence] other species," said Cusenza.


The goal is to complete the project within the next year.  Ultimately, the idea is to develop microarrays with SNPs specific to certain strains that will serve as a rapid detection tools for identification of these strains, and to make the arrays and the underlying information about genetic variation publicly available. "We should identify potentially thousands of single nucleotide polymorphisms which will be really helpful for the Salmonella community," Andersen said.


Perlegen is not disclosing the amount of the NIAID grant that enables this research.


When Curagen spun off its subsidiary, 454, three years ago, the goal was to develop technology that could rapidly sequence genomes. A year ago at the Genome Sequencing and Analysis conference, the Branford, Conn., company revealed that it was going to accomplish this task with a massively parallel approach that sequenced fragments of DNA in a plate containing hundreds of thousands of picoliter-sized solid wells.


This year at GSAC, which was held in Savannah, Ga. between Sunday and Wednesday, Kent Lohman, 454's senior director of molecular sciences, announced that the company has already been able to apply its sequencing technology to rapidly sequence a complete Staphylococcus aureus genome, from start to finished assembly, in a matter of days.


This now-proven ability to rapidly sequence microbial genomes has obvious potential applications in biodefense, according to Richard Begley, 454's CEO. While Begley told GenomeWeb News in an interview today that the company sees the biodefense market as just one among "a variety of markets that we are going to be looking at as we scale up from smaller organisms to larger ones," he emphasized the technology's comparative advantages over other viral or bacterial detection technologies for biodefense applications.


"The biodefense community has been looking a lot at various types of technology for detecting an organism," he said. "But what they do right now, with these portable instruments that fit in a briefcase type thing, [is] they can say 'Is it anthrax?', 'Is it smallpox?', et cetera. So they can tell you the different organism, but they can't tell you what strain of the organism it is. How do you know, if it's anthrax, is it one of the more benign varieties, or was it weaponized? In order to answer that question, you need a second class of instrumentation. And that's what we provide."


Currently, the company's technology can sequence and assemble a virus within less than 36 hours, according to Begley. A single plate in the system can handle at least 2.5mb of 50-bp fragments, and can run the sequencing reactions in about an hour and 45 minutes. But the company has also ramped up to plates of 100-bp fragments, which it can run in 4 hours, and is developing a plate that can do in excess of 10 million bases per hour, said Begley. The company is currently "continuing to improve and streamline" the sample preparation and amplification steps in its plate-based sequencing, Begley said. It is also working with Eugene Myers, the man behind the Celera human genome assembly and now a professor at the University of California, Berkeley, on improving its assembly programs to be able to quickly piece together such short fragments (which are a fraction of the length of Sanger sequencing reads), into a completed genome.


In the hardware area, 454 is miniaturizing the data processing side by using field programmable gate arrays, a standard technology for high-performance computing circuitry, which can do 240 billion transactions per second . "We hope to do the assembly on a single printed.circuit board in a single computer in the instrument," Begley said. 


Although this miniaturization is not being currently driven by the specs of a military contractor, Begley said he believes the system could be put on an aircraft carrier. "I keep kidding our engineering that someday we should be able to put it in the back of a Humvee---which I don't think will happen any time soon," he said. "I think it's more practical to say that we could put these systems at suspicious sites, public health outbreak sites or theaters of war, where we could do the strain analysis."


The company has not yet entered into any contracts with military or government agencies-.and this has been intentional, according to Begley. "The last thing you want to do in the past two years after September 11th is walk in and say you've got this whizzy technology, use me and it will solve all your biodefense problems. That's a stupid thing to do," he said. "We wanted to make sure we were absolutely sure we had something to offer before we approached this community. We are now ready."


The company has made some preliminary phone calls and introduced itself, Begley said. Additionally, it has recently brought several biodefense-public health experts onto its scientific advisory board, including Ian Lipkin of Columbia University and the New York State Department of Health, the principal investigator on one of the NIAID's Regional Centers of Excellence in Biodefense grants; and Jorge Galan of Yale, who studies microbial pathogenesis.


Further on, 454 is looking beyond sequencing microbes, toward what Begley views as another potentially dangerous group of organisms.  "If you want to worry about a public health and biodefense threat, we keep worrying about viruses and bacteria, [but] fungi do 200 billion dollars worth of crop damage every year. There are maybe four million fungi, and only five will be sequenced by the end of this year."  Now, Begley said, "it is taking a whole genome center to do a fungus. I want to be able to do a fungus in a couple of days to a week."

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