Reassociation kinetics-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus
- Equal contributors
1 USDA-ARS, Knipling-Bushland U.S. Livestock Insects Research Laboratory, 2700 Fredericksburg Rd., Kerrville, TX 78028, USA
2 Center for Comparative Genomics, Murdoch University, South St., Perth, Western Australia, 6150, Australia
3 Department of Plant & Soil Sciences and Life Sciences & Biotechnology Institute, Mississippi State University, 117 Dorman Hall, Box 9555, Mississippi State, MS 39762, USA
4 The J. Craig Venter Institute, 9704 Medical Center Drive, Rockville, MD 20850, USA
5 The BecA-ILRI Hub (Biosciences eastern and central Africa - International Livestock Research Institute), PO Box 30709, Nairobi, Kenya
BMC Genomics 2010, 11:374 doi:10.1186/1471-2164-11-374Published: 11 June 2010
The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically problematic. To selectively obtain gene-enriched regions of this tick's genome, Cot filtration was performed, and Cot-filtered DNA was sequenced via 454 FLX pyrosequencing.
The sequenced Cot-filtered genomic DNA was assembled with an EST-based gene index of 14,586 unique entries where each EST served as a potential "seed" for scaffold formation. The new sequence assembly extended the lengths of 3,913 of the 14,586 gene index entries. Over half of the extensions corresponded to extensions of over 30 amino acids. To survey the repetitive elements in the tick genome, the complete sequences of five BAC clones were determined. Both Class I and II transposable elements were found. Comparison of the BAC and Cot filtration data indicates that Cot filtration was highly successful in filtering repetitive DNA out of the genomic DNA used in 454 sequencing.
Cot filtration is a very useful strategy to incorporate into genome sequencing projects on organisms with large genome sizes and which contain high percentages of repetitive, difficult to assemble, genomic DNA. Combining the Cot selection approach with 454 sequencing and assembly with a pre-existing EST database as seeds resulted in extensions of 27% of the members of the EST database.