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Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions

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Abstract
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.
Keywords
ENERGY USE EFFICIENCY, NOVO DNA METHYLATION, EPIGENETIC MUTATION, RESOLUTION MAPS, ARABIDOPSIS, DEMETHYLATION, METHYLTRANSFERASES, TOLERANCE, EPIGENOME, SELECTION, DNA methylation, Reduced representation bisulfite sequencing, RRBS, Oryza sativa, Epiline, Cytosine methylation, Rice, Plant

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MLA
Schmidt, Martin et al. “Plant-RRBS, a Bisulfite and Next-generation Sequencing-based Methylome Profiling Method Enriching for Coverage of Cytosine Positions.” BMC PLANT BIOLOGY 17 (2017): n. pag. Print.
APA
Schmidt, Martin, Van Bel, M., Woloszynska, M., Slabbinck, B., Martens, C., De Block, M., Coppens, F., et al. (2017). Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions. BMC PLANT BIOLOGY, 17.
Chicago author-date
Schmidt, Martin, Michiel Van Bel, Magdalena Woloszynska, Bram Slabbinck, Cindy Martens, Marc De Block, Frederik Coppens, and Maria Van Lijsebettens. 2017. “Plant-RRBS, a Bisulfite and Next-generation Sequencing-based Methylome Profiling Method Enriching for Coverage of Cytosine Positions.” Bmc Plant Biology 17.
Chicago author-date (all authors)
Schmidt, Martin, Michiel Van Bel, Magdalena Woloszynska, Bram Slabbinck, Cindy Martens, Marc De Block, Frederik Coppens, and Maria Van Lijsebettens. 2017. “Plant-RRBS, a Bisulfite and Next-generation Sequencing-based Methylome Profiling Method Enriching for Coverage of Cytosine Positions.” Bmc Plant Biology 17.
Vancouver
1.
Schmidt M, Van Bel M, Woloszynska M, Slabbinck B, Martens C, De Block M, et al. Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions. BMC PLANT BIOLOGY. 2017;17.
IEEE
[1]
M. Schmidt et al., “Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions,” BMC PLANT BIOLOGY, vol. 17, 2017.
@article{8527460,
  abstract     = {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.},
  articleno    = {115},
  author       = {Schmidt, Martin and Van Bel, Michiel and Woloszynska, Magdalena and Slabbinck, Bram and Martens, Cindy and De Block, Marc and Coppens, Frederik and Van Lijsebettens, Maria},
  issn         = {1471-2229},
  journal      = {BMC PLANT BIOLOGY},
  keywords     = {ENERGY USE EFFICIENCY,NOVO DNA METHYLATION,EPIGENETIC MUTATION,RESOLUTION MAPS,ARABIDOPSIS,DEMETHYLATION,METHYLTRANSFERASES,TOLERANCE,EPIGENOME,SELECTION,DNA methylation,Reduced representation bisulfite sequencing,RRBS,Oryza sativa,Epiline,Cytosine methylation,Rice,Plant},
  language     = {eng},
  pages        = {14},
  title        = {Plant-RRBS, a bisulfite and next-generation sequencing-based methylome profiling method enriching for coverage of cytosine positions},
  url          = {http://dx.doi.org/10.1186/s12870-017-1070-y},
  volume       = {17},
  year         = {2017},
}

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