Cot filtration approach for advancing genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus

The size and repetitive nature of the Rhipicephalus microplus genome makes obtaining a full genome sequence fiscally and technically difficult with current technologies. To bridge the gap in sequence information between traditional ESTs and whole genome sequence, we used Cot filtration to selectively obtain gene-enriched regions of this tick's genome. This Cot-filtered DNA was sequenced via 454 pyrosequencing. The sequenced Cot-filtered genomic DNA was assembled with an EST-based gene index of 14,586 unique entries (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=b_microplus) where each EST served as a potential “seed” for assembly and scaffold formation. 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. Current sequence database for R. microplus includes, 175Mb of assembled contigs sequences from Cot-based sequencing for gene enriched regions, three transcriptome library assemblies (21Mb) representing over 33,000 transcripts, BAC-end sequences (7.2Mb), and targeted whole BAC sequence (1.5Mb).