Methylation-sensitive linking libraries enhance gene-enriched sequencing of complex genomes and map DNA methylation domains
AffiliationArizona Genomics Computational Laboratory, BIO5 Institute, University of Arizona, Tucson, Arizona, USA
Arizona Genomics Institute, Department of Plant Sciences, BIO5 Institute, University of Arizona, Tucson, Arizona, USA
Department of Genetics, University of Georgia, Athens, Georgia, USA
Department of Plant Biology, University of Georgia, Athens, Georgia, USA
Plant Genome Initiative at Rutgers, Waksman Institute, Rutgers, The State University of New Jersey, Piscataway, New Jersey, USA
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana, USA
College of Life Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, PR China
Department of Agronomy, Purdue University, West Lafayette, Indiana, USA
Department of Microbial & Plant Genomics, University of Minnesota, St. Paul, MN, USA
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CitationBMC Genomics 2008, 9:621 doi:10.1186/1471-2164-9-621
Rights© 2008 Nelson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0).
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AbstractBACKGROUND:Many plant genomes are resistant to whole-genome assembly due to an abundance of repetitive sequence, leading to the development of gene-rich sequencing techniques. Two such techniques are hypomethylated partial restriction (HMPR) and methylation spanning linker libraries (MSLL). These libraries differ from other gene-rich datasets in having larger insert sizes, and the MSLL clones are designed to provide reads localized to "epigenetic boundaries" where methylation begins or ends.RESULTS:A large-scale study in maize generated 40,299 HMPR sequences and 80,723 MSLL sequences, including MSLL clones exceeding 100 kb. The paired end reads of MSLL and HMPR clones were shown to be effective in linking existing gene-rich sequences into scaffolds. In addition, it was shown that the MSLL clones can be used for anchoring these scaffolds to a BAC-based physical map. The MSLL end reads effectively identified epigenetic boundaries, as indicated by their preferential alignment to regions upstream and downstream from annotated genes. The ability to precisely map long stretches of fully methylated DNA sequence is a unique outcome of MSLL analysis, and was also shown to provide evidence for errors in gene identification. MSLL clones were observed to be significantly more repeat-rich in their interiors than in their end reads, confirming the correlation between methylation and retroelement content. Both MSLL and HMPR reads were found to be substantially gene-enriched, with the SalI MSLL libraries being the most highly enriched (31% align to an EST contig), while the HMPR clones exhibited exceptional depletion of repetitive DNA (to ~11%). These two techniques were compared with other gene-enrichment methods, and shown to be complementary.CONCLUSION:MSLL technology provides an unparalleled approach for mapping the epigenetic status of repetitive blocks and for identifying sequences mis-identified as genes. Although the types and natures of epigenetic boundaries are barely understood at this time, MSLL technology flags both approximate boundaries and methylated genes that deserve additional investigation. MSLL and HMPR sequences provide a valuable resource for maize genome annotation, and are a uniquely valuable complement to any plant genome sequencing project. In order to make these results fully accessible to the community, a web display was developed that shows the alignment of MSLL, HMPR, and other gene-rich sequences to the BACs
this display is continually updated with the latest ESTs and BAC sequences.
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Except where otherwise noted, this item's license is described as © 2008 Nelson et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0).