Source: BAYLOR COLLEGE OF MEDICINE submitted to
GENOME SEQUENCING OF MORAXELLA BOVIS, THE AGENT OF BOVINE KERATOCONJUNCTIVITIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0200101
Grant No.
2004-35600-14577
Project No.
TEXR-2004-04989
Proposal No.
2004-04989
Multistate No.
(N/A)
Program Code
23.2
Project Start Date
Jul 15, 2004
Project End Date
Jul 14, 2008
Grant Year
2004
Project Director
Highlander, S. K.
Recipient Organization
BAYLOR COLLEGE OF MEDICINE
(N/A)
HOUSTON,TX 77030
Performing Department
(N/A)
Non Technical Summary
Infectious bovine keratoconjunctivitis (IBK), or "pinkeye" is a significant and highly contagious ocular infection of cattle. The primary causative agent is Moraxella bovis, a gram-negative coccobacillus that is difficult to control by vaccination or antibiotic treatment. Though IBK is rarely fatal, it causes considerable losses to the cattle and dairy industries because of decreased weight gain, decreased milk production, devaluation because of eye disfigurement and because of the high cost of treatment of the disease. A 1993 study estimated that these losses totaled over $150 million per year, though losses as high as $200 million per year have also been estimated. In 1996, IBK was second only to scours and diarrhea as the most common affliction of unweaned calves in the United States; it was the most prevalent disease in breeding beef females. Determining the genome sequence of Moraxella bovis should aid in development of vaccines to prevent, or therapies to prevent bovine keratoconjunctivitis.
Animal Health Component
100%
Research Effort Categories
Basic
80%
Applied
10%
Developmental
10%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
31140101100100%
Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;
Goals / Objectives
The objectives of this project are to determine the complete genomic sequence of Moraxella bovis Epp63, assemble the sequence and annotate the genes contained within the sequence.
Project Methods
We propose to determine the ca. 2 Mb whole genome sequence of Moraxella bovis strain Epp63. This will be accomplished by a combination of random shotgun sequencing of small insert (2 kb) libraries (SIC), medium (10 kb) insert clones (MIC) and large insert (30 kb) libraries (LIC). All DNA sequencing will be performed at the Baylor College of Medicine Human Genome Sequencing Center (HGSC). Our strategy involves performing paired-end sequencing of SICs to 8-fold coverage, followed by paired-end sequencing of about 1600 MICs. Paired-end analysis will allow creation of assembled scaffolds that can further be closed into a contiguous sequence through a combination of end sequencing of LICs and overgo hybridization to link LICs. Remaining gaps will be closed by PCR and primer-walking. Sequence quality will be assessed using the phred base-calling program and only sequences with phred scores of 20 or higher will be included in assemblies. Final assembly will be verified by fingerprinting of LICs that form a contiguous tiling path on the genome. Sequence information will be made available on a website at the HGSC. We will also hold two sequencing workshops where Moraxella researchers can share sequence and sequence analysis tools; this will also lead to development of a community-based annotation program.

Progress 07/15/04 to 07/14/08

Outputs
OUTPUTS: We created and sequenced small insert libraries of Moraxella bovis Epp63 genomic DNA by the Sanger method to 2X coverage. We added 20X 454 GS20 and 454 paired end sequencing, and a Solexa run that provided 32X sequence coverage. The genome was originally assembled into 64 scaffolds with a genome size estimated at 2.8 Mb. This is 800 kilobases (kb) larger than our earlier estimate. Autofinish was performed and nearly twenty rounds of manual finishing were performed. The current assembly consists of 235 contigs in ten scaffolds with an N50 of 56,408 bases. The longest scaffold is 450,825 bp long and the shortest is 120 bp in length. A 27 kb probable conjugal plasmid was also sequenced, assembled and annotated. We had hoped to provide a single scaffold with all contigs ordered and oriented, but despite deep coverage, heroic finishing efforts and expenditure of local funds, we were unable to order the final ten scaffolds. Gene predictions have been made and the genome is now ready for semi-automated annotation. Though the project period has ended, we are committed to completing the annotation and publication of our findings. PARTICIPANTS: Individuals 1. PIs: Sarah Highlander and George Weinstock: supervised the project 2. Xiang Qin: Did assemblies Collaborators: At BCM: Richard Gibbs: Director of the Human Genome Research Center Donna Muzny: supervised the sequencing and finishing Lynne Nazareth and Sandra Lee: created libraries for all sequencing Shannon Dugan and Yan Ding: worked on finishing Outside BCM: Akif Uzman, University of Houston Downtown Anthony Confer, University of Calgary, CA Ian Peak, Griffith University, AUS Lisle George, University of California, Davis Richard Strugnell, University of Melboure, AUS Training Annotation will train graduate students and undergraduate students in microbial genomics and microbial physiology TARGET AUDIENCES: Target audiences are individuals interested in the pathogenesis of the Moraxellaceae and those interested in developing vaccines and therapeutics for Moraxella disease. PROJECT MODIFICATIONS: As mentioned in Outcomes/Impacts, we made a radical change in our sequencing pipeline, moving the proposed 100% Sanger sequencing to a mixture of Next Generation sequencing technologies, including 454 and Solexa sequencing.

Impacts
During this project, we made radical changes in our sequencing pipeline. The original proposal aimed to sequence M. bovis using Sanger sequencing alone. We now sequence microbes using a combination of 454 and Solexa sequencing. We use the 454 FLX for fragment and paired-end sequencing and layer that with the short Solexa reads. The only Sanger sequencing that is done is for finishing activities. Assembly is also done in a different way. Newbler and Mosaik are used, instead of phrap, for short read assemblies.

Publications

  • No publications reported this period


Progress 08/01/06 to 07/31/07

Outputs
OUTPUTS: Sequencing was accomplished using a combination of random shotgun sequencing of small insert (2 kb) libraries, 454 Life Sciences pyrosequencing and Solexa sequencing. All DNA sequencing was performed at the Baylor College of Medicine Human Genome Sequencing Center (HGSC). Remaining gaps are being closed by manual PCR and primer walking. Using a combination of low coverage (2X) Sanger sequencing and high coverage (20X) 454 sequencing, the genome was assembled into 64 scaffolds with a genome size estimated at 2.7 Mb. This was 700 kb larger than our earlier estimate. The Solexa run yielded 2,440.298 reads with an average sequence length of 36 bp for 32X coverage. The current assembly consists of 368 contigs in thirteen scaffolds with an N50 of 1,560,924. The scaffold length is 2.75 Mb. A 27 kb probable conjugal plasmid has been sequenced, assembled and annotated. Annotation will begin shortly. PARTICIPANTS: PI Highlander: supervised the entire project PI Weinstock: supervised the sequencing and assembly collaborator Muzny: supervised the finishing PROJECT MODIFICATIONS: Included next-generation sequencing techniques to facilitate closure of the genome.

Impacts
Once the annotation is complete, we will have a gene list, which will allow us to make predictions of genes and proteins involved in virulence of Moraxella bovis. This should aid those interested in producing vaccines to prevent and therapeutics to treat bovine keratoconjunctivitis. This could have an impact on the prevalence of this disease in dairy cows and cattle.

Publications

  • No publications reported this period


Progress 10/01/05 to 09/30/06

Outputs
Infectious bovine keratoconjunctivitis (IBK), or "pinkeye" is a significant and highly contagious ocular infection of cattle. The primary causative agent is Moraxella bovis, a gram-negative coccobacillus that is difficult to control by vaccination or antibiotic treatment. Though IBK is rarely fatal, it causes considerable losses to the cattle and dairy industries because of decreased weight gain, decreased milk production, devaluation because of eye disfigurement and because of the high cost of treatment of the disease. A 1993 study estimated that these losses totaled over $150 million per year, though losses as high as $200 million per year have also been estimated. In 1996, IBK was second only to scours and diarrhea as the most common affliction of unweaned calves in the United States; it was the most prevalent disease in breeding beef females. Sequencing has been accomplished using a combination of random shotgun sequencing of small insert (2 kb) libraries and 454 Life Sciences pyrosequencing. Remaining gaps will be closed by PCR and primer walking. Using a combination of low coverage (2X) Sanger sequencing and high coverage (20X) 454 sequencing, the genome has been assembled into 64 scaffolds with a genome size estimated at 2.7 Mb. This is 700 kb larger than our earlier estimate. A 27 kb probable conjugal plasmid has been sequenced, assembled and annotated. Once the chromosomal sequence is closed we will begin manual annotation of the genome.

Impacts
With respect to intellectual merit, this project will provide information on potential pathogenic features of an important bovine pathogen and should eventually lead to development of therapeutics to treat, or vaccines to prevent, M. bovis disease. This will be the first complete Moraxella species sequenced so the genome information may also be of interest to those interested in M. cattharalis, a cause of otitis media in humans. Though not a defined goal of this project, an additional intellectual impact that may result is the development or improvement of genome assembly and analysis tools.

Publications

  • No publications reported this period