Integration of cot analysis, DNA cloning, and high-throughput sequencing facilitates genome characterization and gene discovery

Abstract

Cot-based sequence discovery represents a powerful means by which both low-copy and repetitive sequences can be selectively and efficiently fractionated, cloned, and characterized. Based upon the results of a Cot analysis, hydroxyapatite chromatography was used to fractionate sorghum (Sorghum bicolor) genomic DNA into highly repetitive (HR), moderately repetitive (MR), and single/low-copy (SL) sequence components that were consequently cloned to produce HRCot, MRCot, and SLCot genomic libraries. Filter hybridization (blotting) and sequence analysis both show that the HRCot library is enriched in sequences traditionally found in high-copy number (e.g., retroelements, rDNA, centromeric repeats), the SLCot library is enriched in low-copy sequences (e.g., genes and "nonrepetitive ESTs"), and the MRCot library contains sequences of moderate redundancy. The Cot analysis suggests that the sorghum genome is approximately 700 Mb (in agreement with previous estimates) and that HR, MR, and SL components comprise 15%, 41%, and 24% of sorghum DNA, respectively. Unlike previously described techniques to sequence the low-copy components of genomes, sequencing of Cot components is independent of expression and methylation patterns that vary widely among DNA elements, developmental stages, and taxa. High-throughput sequencing of Cot clones may be a means of "capturing" the sequence complexity of eukaryotic genomes at unprecedented efficiency.