Abstract
BackgroundThe 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.ResultsThe 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.ConclusionCot 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.
Highlights
The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically problematic
The library consists of 46,080 clones, providing ~0.8 X coverage of the R. microplus genome
BACs 77-J9 and 74-F12 were selected at random, BAC 129-N14 was selected based on its hybridization to a probe from a pyrethroid-metabolizing carboxylesterase (CzEst9; [10]), while BACs 66-M7 and 77-G20 were selected based on hybridization to a mixed probe containing equal amounts of cDNAs encoding a putative CYP41 cytochrome P450 family member [18] and a putative acetylcholinesterase AChE1 [19]
Summary
The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically problematic. Rhipicephalus (Boophilus) microplus, the tropical/southern cattle tick, is a livestock ectoparasite which has negatively impacted the cattle industry throughout the world. This tick is a vector for the pathogenic organisms which cause bovine babesiosis and anaplasmosis. Annual economic losses attributable to R. microplus infestations have been estimated in Brazil and Australia to be approximately $2 billion [1] and over $100 million [2], respectively. S. cattle industry of over $130 million in the early 1900s, equivalent to approximately $3 billion in 2009 dollars [3].
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