Improved Chemistry and Software Provides Higher Accuracy Single
Molecule Reads and Longer Readlengths to
The data provided to the public domain includes a complete assembly of the German outbreak strain, alignment to assemblies from other outbreak isolates, and sequences for 11 related Enteroaggregative E. coli strains. The project demonstrates the ability to produce a PacBio-only de novo assembly for a complex microbial pathogen, and the power of rapid sequencing of multiple genomes with the PacBio RS to elucidate the evolutionary history of a pathogenic microbe. A summary of the project appears on the company's website at http://blog.pacificbiosciences.com.
The Pacific Biosciences scientific team, led by Chief Scientific Officer
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Karen Angeliki Krogfelt, Ph.D., Professor, Head of Unit,
Gastrointestinal Infections, Statens Serum Institut (SSI),
Denmark -
Flemming Scheutz , Ph.D., Head of theWHO Collaborating Centre for Reference and Research on Escherichia and Klebsiella, SSI,Denmark
In the U.S.:
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James P. Nataro , M.D., Ph.D., Professor and Chair, Pediatrics,University of Virginia School of Medicine -
David A. Rasko , Ph.D., Assistant Professor,University of Maryland School of Medicine ,Institute for Genome Sciences andDepartment of Microbiology and Immunology -
Nadia Boisen , Ph.D., Research Scientist,Department of Pediatrics ,University of Virginia School of Medicine -
Matthew K. Waldor , M.D., Ph.D., Professor of Medicine atHarvard Medical School , Brigham and Women's Hospital, and HMMI
"Using samples provided by our collaborators, we rapidly sequenced each strain using a standard PacBio RS protocol that took on average less than eight hours from sample preparation to sequencing results," said Dr. Schadt. "The ability to sequence the outbreak strain with reads averaging 2,900 base pairs and our longest reads at over 7,800 bases, combined with our circular consensus sequencing to achieve high single molecule accuracy with a mode accuracy distribution of 99.9%, enabled us to complete a PacBio-only assembly without having to construct specialized fosmid libraries, perform PCR off the ends of contigs, or other such techniques that are required to get to similar assemblies with second generation DNA sequencing technologies."
Dr. Krogfelt commented: "These high quality data will provide scientists with more information about the genomic features of this strain that could provide new markers for predicting the higher degree of pathogenicity we are seeing with this outbreak. A more comprehensive evolutionary view of this pathogen may also help identify markers for antibiotic drug resistance that could be used in the future should other related strains emerge. The complexity of this case proves that international collaborations and communications are important in the achievement of detailed scientific information."
The data are available for the bioinformatics community at the PacBio
developer's network (DevNet) web site (www.pacbiodevnet.com),
where a suite of open source tools and other resources designed for SMRT
sequence data are available to analyze the information. The data have
also been submitted to the
While not involved with the current
- An average of 2,700 base pair reads, with 5% of the reads achieving 5,100+ base pairs
- Up to 90 megabases of mappable data per SMRT Cell
- 1,350 base pair reads with 2X circular consensus sequencing single molecule accuracy of 93%
For more information about Pacific Biosciences, please visit www.pacificbiosciences.com. You can also follow the company on twitter www.twitter.com/pacbio.
About Pacific Biosciences
Pacific Biosciences' mission is to transform the way humankind acquires, processes and interprets data from living systems through the design, development and commercialization of innovative tools for biological research. The company has developed a novel approach to studying the synthesis and regulation of DNA, RNA and proteins. Combining recent advances in nanofabrication, biochemistry, molecular biology, surface chemistry and optics, Pacific Biosciences has created a powerful technology platform called single molecule, real-time, or SMRT™, technology. SMRT technology enables real-time analysis of biomolecules with single molecule resolution, which has the potential to transform the understanding of biological systems by providing a window into these systems that has not previously been open for scientific study.
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