Reassociation kinetics-based approach for partial genome sequencing of the cattle tick, Rhipicephalus (Boophilus) microplus

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Maps of the sequenced BACs. Each BAC is represented on a number line marked in kb. Arrows above the number line represent where Genscan search found GenBank sequences that had significant sequence similarity (e < 0.001) to regions of each BAC. The direction of transcription (5'-3' positive direction shown as left to right) is indicated. BAC represented are a) BM-66-M7; b) BM-77-G20; c) BM-129-N14; d) BM-77-J9; and e) BM-74-F12. We also indicate the identity and e-value for the statistically significant Blast hits (e < 0.001) for the gene predictions indicated above the number line for each BAC. The complete Genscan results are in Additional Files 1 and 2.

Figure 2

NUCmer analysis of the BACs.NUCmer Version 3.06 was used to plot all 5 BAC sequences. Matches unique in the reference sequence but not necessarily unique in the query are shown in blue. All other matches are shown in red.

Figure 3

Distribution of the BmiGI member extensions resulting from reassembly with the Cot-selected genomic DNA sequences. The range of length of the extension is noted on the x-axis in bp and the count of the number of members of BmiGI Version 2.1 that were extended within a specific range is noted on the y-axis.