Recipient Organization
UNIV OF OKLAHOMA HEALTH SCIENCES CTR
1100 N. LINDSAY
OKLAHOMA CITY,OK 73104
Performing Department
MICROBIOLOGY & IMMUNOLOGY
Non Technical Summary
In this application, we propose to determine the genome sequence of the important veterinary pathogen Actinobacillus pleuropneumoniae (Ap), a causative agent of severe respiratory disease in swine. Ap strains vary in their virulence for swine, and in the US, serotype 1 strains are among the most virulent. We therefore propose to sequence to completion the genome of Ap strain 4074, which has been widely used for experimental studies on serotype 1 disease. In future studies, we also will perform low-density sequencing on the genome of Ap serotype 7 strain WF83. This low-density genome sequence will then be compared to the sequence of the serotype 1 strain, to begin to understand the genomic basis underlying the differences in virulence between these Ap serotypes. The sequence data will be publicly available on our web site (http://micro-gen.ouhsc.edu) and will be updated at monthly intervals.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Specific aim 1: We will determine the complete nucleotide sequence and annotate the genome of Ap serotype 1 strain 4074.
Project Methods
For full genome sequencing, we will focus on Ap strain 4047, the type strain for serotype 1 organisms. This strain is widely used for studies on the most common serotype found to cause infections in the US. First, we will construct at least one plasmid library in pUC18, with a 2-4kb average insert size, and shotgun sequence this library to approximately 8-fold coverage. During the initial phases of this shotgun sequencing, the data will be carefully screened to ensure that the library is sufficiently random; otherwise, additional libraries will be built. For the 2.4Mb Ap genome, this shotgun sequencing phase will require 38,400 sequences reads, according to Lander-Waterman calculations. Since our colony-to-sequence read efficiency has been approximately 85% (data not shown), this indicates that we will have to pick about 45,176 colonies to obtain 38,400 reads of sufficient quality for assembly. Simultaneously, we will construct a cosmid-sized (ca. 40kb) large-insert
library in pBeloBac11 and end-sequence approximately 1000 clones. This BAC data will be assembled with the shotgun data from the pUC library(s), and will form a backbone upon which the shotgun data can be organized for final closure and annotation. The data will be assembled using Phred/Phrap, and the sequence viewed in Consed for assessing data quality and design of closure experiments. Early in the shotgun sequencing phase (ca.3-4-fold coverage), we will begin to resolve problems caused by repeats by using Consed to identify putative repeat regions, and designing combinatorial PCR experiments to isolate these repeats on PCR amplicons. These amplicons will be primer-walked simultaneously with the latter portion of the shotgun sequencing phase. Final closure will rely on PCR and additional primer walking to close gaps, with primer-directed finishing employed to raise sequence quality in low-coverage regions of the genome. The location and exact sequence of repeats such as the rrn
operons will be confirmed by isolating PCR fragments that contain each repeat in its entirety, followed by primer walking across the PCR product.