ARS | Publication request: Reassociation kinetics analysis-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus

Submitted to: Biomed Central (BMC) Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 11, 2010
Publication Date: June 11, 2010
Citation: Guerrero, F., Moolhuijzen, P., Peterson, D.G., Bidwell, S., Caler, E., Appels, R., Bellgard, M., Nene, V.M., Djikeng, A. 2010. Reassociation kinetics analysis-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus. Biomed Central (BMC) Genomics. 11:Article 374).

Interpretive Summary: The cattle tick, Rhipicephalus (Boophilus) microplus, has a genome over 2.4 times the size of the human genome, and with over 70% of repetitive DNA, this genome would prove very costly to sequence at today's prices and almost impossible to assemble using current technology. However, the current cattle tick gene database is not complete and existing entries are often missing nucleotides which encode the N-terminal regions of tick proteins. Thus, to efficiently obtain more sequence from tick coding regions, a procedure called Cot-filtration was used to reduce the repetitive DNA fraction from a sample of cattle tick genomic DNA. Subsequently, this DNA was sequenced using pyrosequencing FLX 454 methodology. The new sequence information was used to assemble a new cattle tick gene database, with over 26% of the pre-existing gene entries extended with the 454-acquired sequence. Combining the Cot-filtration approach with 454 sequencing and assembly onto traditional EST databases as seeds was found to be an efficient bridge between EST-based sequencing and a full genome sequencing project.

Technical Abstract: The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence unlikely with current technology and costs. Thus, Cot filtration/selection techniques were used to reduce the repetitive fraction of the tick genome and enrich for gene-containing regions. The Cot-selected genomic DNA was sequenced using 454 FLX pyrosequencing and assembled with an EST-based gene index of 14,587 unique entries serving as seed sequences. The new sequence assembly extended the lengths of 3,913 of the 14,587 gene index entries. Over half of the extensions corresponded to extensions of over 30 amino acids. For a survey of occurrence of repetitive elements in the tick genome, the completed sequences of 5 BAC clones were also determined. Sequences corresponding to many types of both Class I and II transposable elements were found, however, the Cot selection process reduced the copy number to considerably less that what was expected in non-selected DNA. The Cot selection protocol 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 onto traditional EST databases as seeds resulted in extensions of a significant number of EST-based contigs.