2012 Annual Report
1a.Objectives (from AD-416):
The U.S. cattle industry's annual losses attributable to Boophilus ticks were estimated in 1906 to be $130,500,000 and led to a Boophilus eradication program. Due to the prevalence of pathogen-infected Boophilus ticks in Mexico, the eradication status is maintained by the USDA-APHIS Cattle Fever Tick Eradication Program using a 500-mile "buffer zone" along the U.S.-Mexico border with mandatory acaricide treatment of livestock prior to importation into the U.S. Annual costs for this program are over $4 million.
Rhipicephalus (Boophilus) microplus has rapidly developed acaricide resistance in many countries, and the resistance phenotype extends to several chemical classes. The development of R. microplus populations resistant to multiple acaricides is a major risk factor for re-establishment of this tick in the U.S. Genomic sequence data for R. microplus should enhance the potential for the identification of biological targets for developing novel chemistries to effect tick control strategies complementing current control methods. The feasibility of controlling R. microplus through use of a recombinant vaccine was shown by the commercial release of such a vaccine in 1994. The limitations of the current vaccine are well known; but experimentally, it has been shown that a vaccine can have efficacy equivalent to that of a chemical acaricide. Improvement of the existing vaccine and development of novel, more effective vaccines demands a better understanding of tick biology and an efficient identification of cattle tick antigens. These aims would be greatly facilitated by a cattle tick genome sequence.
The R. microplus genome size was determined to be approximately 7.1 X 109 bp and consists of 30% unique, 38% moderately repetitive, 31% highly repetitive, and 0.82% foldback DNA. An approximate 3X coverage BAC library is available in our laboratory. Our group has also generated 12,000 BAC end sequences, 15 whole BAC sequences, a gene index of approximately 28,000 unique putative coding regions, and several runs of 454-based genomic DNA sequencing which produced 1.8 X 109 bp (0.25X coverage). The objective of this agreement is to facilitate the assembly and annotation of the cattle tick genome and transcriptome by acquistion of sequence data from the next-generation Illumina sequencing platform and the third generation Pacific Biosciences sequencing platform. Both approaches will provide new cattle tick sequence information that will ensure complete coverage of the cattle tick transcriptome, provide cattle tick genome sequences of sufficient length to traverse many of the problematic highly repetitive regions of the cattle tick genome sequence, and should greatly increase the contig length of sequences in the cattle tick genome sequence database. Additionally, the collaborator's expertise with Illumina and Pacific Biosciences technologies will be essential and their bioinformatics staff will be critical in analysis of the data.
1b.Approach (from AD-416):
The cattle tick genome sequencing has advanced to the stage where novel approaches are necessary to advance the project. The less complex regions of the cattle tick genome and the most accessible genes of the transcriptome have been sequenced using Sanger and 454 technology. Next-generation Illumina and Third generation Pacific Biosciences sequencing approaches are the only methodologies that can sequence through the difficult regions of the cattle tick genome that have yet to be attempted.
1. Illumina RNA-Seq approach will be used for deep sequencing transcriptome of cattle tick. This approach is estimated to acquire approximately 95% of the transcribed genes of the organism. Selected organs could be targeted by this approach, with the Gene's Organ a likely tissue of interest. Bioinformatic analysis will also be required to assemble and annotate this data.
2. Quantitative transcriptomics is available through the Illumina RNA-seq platform and will be used to identify quantitative differences in tick gene expression in response to feeding upon a bovine host. Specialized bioinformatics analysis will be required for quantitation of the RNA-seq data.
3. Pacific Biosciences Third Generation sequencing technology will be used for library synthesis and SMRT cell protocol optimized for long read sequencing. Specialized bioinformatic analysis will be required for interpretation, assembly and annotation of this dataset.
4. Extensive computational resources are required to accomplish these analyses.
A pilot study has shown that 3rd generation sequencing technology will produce long sequence reads that can be used to assemble the genome of the cattle tick. Deep sequencing of the cattle tick transcriptome has been utilized to study changes in gene expression of adult cattle ticks during the process of feeding upon cattle.