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Title: Application of combined SMRT and long-read pyrosequencing to produce reference genome sequences of bacteria associated with respiratory disease outbreaks in beef cattle

item Harhay, Gregory
item McVey, David
item KITZMILLER, AARON - Bio Team Inc
item CLAMP, MICHELE - Bio Team Inc
item OSBORNE, BRIAN - Bio Team Inc
item DWAN, CHRIS - Bio Team Inc
item ZHANG, JIESHENG - Bio Team Inc
item SISNEROS, NICK - Pacific Biosciences Inc
item WANG, SUSANA - Pacific Biosciences Inc
item PELUSO, PAUL - Pacific Biosciences Inc
item Bono, James - Jim
item Chitko-Mckown, Carol
item Clawson, Michael - Mike
item Heaton, Michael - Mike
item Harhay, Dayna
item Smith, Timothy - Tim

Submitted to: Advances in Genome Biology and Technology
Publication Type: Abstract Only
Publication Acceptance Date: 12/7/2011
Publication Date: 2/15/2012
Citation: Harhay, G.P., Mcvey, D.S., Kitzmiller, A., Clamp, M., Osborne, B., Dwan, C., Zhang, J., Sisneros, N., Wang, S., Peluso, P., Bono, J.L., Chitko Mckown, C.G., Clawson, M.L., Heaton, M.P., Harhay, D.M., Smith, T.P.L. [abstract]. 2012. Application of combined SMRT and long-read pyrosequencing to produce reference genome sequences of bacteria associated with respiratory disease outbreaks in beef cattle. Advances in Genome Biology and Technology, February 15-18, 2012, Marco Island, Florida. Poster No. 129.

Interpretive Summary:

Technical Abstract: The effectiveness of comparing complete genomes for elucidating mechanisms of virulence in pathogenic organisms has been demonstrated recently in foodborne and waterborne human disease outbreaks. We built upon this concept to investigate virulence mechanisms in bovine respiratory disease complex (BRDC), the most costly (greater than or equal to USD 1 billion/year) disease affecting cattle during beef production in the United States. BRDC is an illness caused by an array of viral and/or microbial agents. The most severe manifestations of the disease appear to involve immune suppression by viral infection, followed by lung invasion from a normally commensal organism, Mannheimia haemolytica. Some strains of M. haemolytica appear more aggressive and communicable than others and, thus, responsible for increased disease severity, economic losses and a reduction in animal well-being. Prior to the start of our project, no reference sequence or scaffold spanning the 2.5-Mb M. haemolytica chromosome was available, so we developed an approach to generate them for different strains with a known range of virulence. These reference sequences or scaffolds are required to compare the chromosomes and to analyze them within the context of strain antibiotic resistance, metabolic phenotypes and known virulence. Using the Celera assembler and associated software, this study demonstrates that single molecule real-time sequencing (SMRT) and 454 reads can be combined to generate robust hybrid assemblies where longer SMRT reads identify mis-assemblies and span gaps in 454 only assemblies, while 454 reads improve the base call confidence in SMRT reads. Our automated genome assembly and annotation pipeline facilitates the routine comparison of genomes that have been uniformly assembled and annotated. This approach was demonstrated with the sequencing, assembly and analysis of seven strains of M. haemolytica and four of the closely related Bibersteinia trehalosi bacteria. USDA is an equal opportunity provider and employer.