Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions
| Autor: | Marc De Block, Cindy Martens, Mieke Van Lijsebettens, Martin Schmidt, Bram Slabbinck, Michiel Van Bel, Magdalena Woloszynska, Frederik Coppens |
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| Jazyk: | angličtina |
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
SELECTION NOVO DNA METHYLATION Bisulfite sequencing Cytosine methylation Oryza sativa Plant Science Biology DNA sequencing DEMETHYLATION EPIGENOME EPIGENETIC MUTATION 03 medical and health sciences chemistry.chemical_compound Cytosine Reduced representation bisulfite sequencing ENERGY USE EFFICIENCY lcsh:Botany Sulfites Methylated DNA immunoprecipitation TOLERANCE 2. Zero hunger Genetics DNA methylation RRBS Methodology Article Biology and Life Sciences food and beverages RESOLUTION MAPS Oryza METHYLTRANSFERASES Plant DNA Restriction Enzymes ARABIDOPSIS lcsh:QK1-989 Bisulfite Epiline 030104 developmental biology chemistry Genetic Techniques Illumina Methylation Assay Rice Genome Plant |
| Zdroj: | BMC Plant Biology, Vol 17, Iss 1, Pp 1-14 (2017) BMC Plant Biology BMC PLANT BIOLOGY |
| ISSN: | 1471-2229 |
| DOI: | 10.1186/s12870-017-1070-y |
| Popis: | Background Cytosine methylation in plant genomes is important for the regulation of gene transcription and transposon activity. Genome-wide methylomes are studied upon mutation of the DNA methyltransferases, adaptation to environmental stresses or during development. However, from basic biology to breeding programs, there is a need to monitor multiple samples to determine transgenerational methylation inheritance or differential cytosine methylation. Methylome data obtained by sodium hydrogen sulfite (bisulfite)-conversion and next-generation sequencing (NGS) provide genome-wide information on cytosine methylation. However, a profiling method that detects cytosine methylation state dispersed over the genome would allow high-throughput analysis of multiple plant samples with distinct epigenetic signatures. We use specific restriction endonucleases to enrich for cytosine coverage in a bisulfite and NGS-based profiling method, which was compared to whole-genome bisulfite sequencing of the same plant material. Methods We established an effective methylome profiling method in plants, termed plant-reduced representation bisulfite sequencing (plant-RRBS), using optimized double restriction endonuclease digestion, fragment end repair, adapter ligation, followed by bisulfite conversion, PCR amplification and NGS. We report a performant laboratory protocol and a straightforward bioinformatics data analysis pipeline for plant-RRBS, applicable for any reference-sequenced plant species. Results As a proof of concept, methylome profiling was performed using an Oryza sativa ssp. indica pure breeding line and a derived epigenetically altered line (epiline). Plant-RRBS detects methylation levels at tens of millions of cytosine positions deduced from bisulfite conversion in multiple samples. To evaluate the method, the coverage of cytosine positions, the intra-line similarity and the differential cytosine methylation levels between the pure breeding line and the epiline were determined. Plant-RRBS reproducibly covers commonly up to one fourth of the cytosine positions in the rice genome when using MspI-DpnII within a group of five biological replicates of a line. The method predominantly detects cytosine methylation in putative promoter regions and not-annotated regions in rice. Conclusions Plant-RRBS offers high-throughput and broad, genome-dispersed methylation detection by effective read number generation obtained from reproducibly covered genome fractions using optimized endonuclease combinations, facilitating comparative analyses of multi-sample studies for cytosine methylation and transgenerational stability in experimental material and plant breeding populations. Electronic supplementary material The online version of this article (doi:10.1186/s12870-017-1070-y) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
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