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Q-RRBS: a quantitative reduced representation bisulfite sequencing method for single-cell methylome analyses.

ABSTRACT: Reduced representation bisulfite sequencing (RRBS) is a powerful method of DNA methylome profiling that can be applied to single cells. However, no previous report has described how PCR-based duplication-induced artifacts affect the accuracy of this method when measuring DNA methylation levels. For quantifying the effects of duplication-induced artifacts on methylome profiling when using ultra-trace amounts of starting material, we developed a novel method, namely quantitative RRBS (Q-RRBS), in which PCR-induced duplication is excluded through the use of unique molecular identifiers (UMIs). By performing Q-RRBS on varying amounts of starting material, we determined that duplication-induced artifacts were more severe when small quantities of the starting material were used. However, through using the UMIs, we successfully eliminated these artifacts. In addition, Q-RRBS could accurately detect allele-specific methylation in absence of allele-specific genetic variants. Our results demonstrate that Q-RRBS is an optimal strategy for DNA methylation profiling of single cells or samples containing ultra-trace amounts of cells.

SUBMITTER: Wang K

PROVIDER: S-EPMC4622980 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCytosine 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.MethodsWe 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.ResultsAs 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.ConclusionsPlant-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.

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Q-RRBS: a quantitative reduced representation bisulfite sequencing method for single-cell methylome analyses.

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