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Methods

Gene enrichment in maize with hypomethylated partial restriction (HMPR) libraries

¹ Department of Biological Sciences, Purdue University, West Lafayette, Indiana 47907, USA ² Genomics Core Facility, Purdue University, West Lafayette, Indiana 47907, USA ³ Department of Genetics, University of Georgia, Athens, Georgia 30602, USA

A new technology was developed to assist gene-enrichment sequencing of any complex plant genome, employing maize as the test organism. Hypomethylated partial restriction (HMPR) libraries were constructed by using independent partial digestions with methylation-sensitive restriction enzymes HpaII (5'-CCGG-3') and HpyCH4IV (5'-ACGT-3'). Fragments of 1-4 kb were purified and cloned, followed by sequence analysis of >2000 clones from 10 separate libraries. Organellar clones comprised 10% of each library but were useful in showing that no chimeric clones were generated and that digestion efficiencies were 10%-25% in different libraries. Four separate HMPR libraries, analyzed in detail, exhibited very similar degrees of gene enrichment and repeat depletion. Known gene homologies were found in 25% of the HMPR clones, compared with <4% in clones from a fully random set of unfiltered maize shotgun sequences. This six- to sevenfold enrichment for genes compares favorably with the best previous gene enrichment techniques in maize, High Cot analysis and methylation filtration. Compared with High Cot and methylation filtration, HMPR is exceptional in depleting retrotransposons' content to the lowest level yet observed (<5%, compared with >70% for unfiltered maize sequences) and in providing an unmatched enrichment for the "unknown" sequences that contain promoters, introns, and other gene-adjacent regions.