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A Practical Guide to the Measurement and Analysis of DNA Methylation.

ABSTRACT: DNA methylation represents a fundamental epigenetic mark that is associated with transcriptional repression during development, maintenance of homeostasis, and disease. In addition to methylation-sensitive PCR and targeted deep-amplicon bisulfite sequencing to measure DNA methylation at defined genomic loci, numerous unsupervised techniques exist to quantify DNA methylation on a genome-wide scale, including affinity enrichment strategies and methods involving bisulfite conversion. Both affinity-enriched and bisulfite-converted DNA can serve as input material for array hybridization or sequencing using next-generation technologies. In this practical guide to the measurement and analysis of DNA methylation, the goal is to convey basic concepts in DNA methylation biology and explore genome-scale bisulfite sequencing as the current gold standard for assessment of DNA methylation. Bisulfite conversion chemistry and library preparation are discussed in addition to a bioinformatics approach to quality assessment, trimming, alignment, and methylation calling of individual cytosine residues. Bisulfite-converted DNA presents challenges for standard next-generation sequencing library preparation protocols and data-processing pipelines, but these challenges can be met with elegant solutions that leverage the power of high-performance computing systems. Quantification of DNA methylation, data visualization, statistical approaches to compare DNA methylation between sample groups, and examples of integrating DNA methylation data with other -omics data sets are also discussed. The reader is encouraged to use this article as a foundation to pursue advanced topics in DNA methylation measurement and data analysis, particularly the application of bioinformatics and computational biology principles to generate a deeper understanding of mechanisms linking DNA methylation to cellular function.

SUBMITTER: Singer BD

PROVIDER: S-EPMC6775954 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDNA methylation is a conserved epigenetic mark that controls genome stability, development and environmental responses in many eukaryotes. DNA methylation can be guided by non-coding RNAs that include small interfering RNAs and scaffold RNAs. Although measurement of DNA methylation and regulatory non-coding RNAs is desirable for many biologists who are interested in exploring epigenetic regulation in their areas, conventional methods have limitations and are technically challenging. For instance, traditional siRNA detection through RNA hybridization requires relatively large amount of small RNAs and involves radioactive isotopes. An alternative approach is RT-qPCR that employs stem loop primers during reverse transcription; however, it requires a prerequisite that the exact sequences of siRNAs should be known.ResultsBy using the model organism Arabidopsis thaliana, we developed an easy-to-follow, integrative procedure for time-efficient, quantitative measurement of DNA methylation, small interfering RNAs, and scaffold RNAs. Starting with simplified nucleic acid manipulation, we examined DNA methylation levels by using Chop PCR (methylation-sensitive enzyme digestion followed by PCR), which allowed for fast screening for DNA methylation mutants without the need of transgenic reporters. We deployed a simple bioinformatics method for mining published small RNA databases, in order to obtain the nucleotide (nt) sequences of individual 24nt siRNAs within the regions of interest. The protocol of commercial TaqMan Small RNA Assay was subsequently optimized for reliable quantitative detection of individual siRNAs. We used nested qPCR to quantify scaffold RNAs that are of low abundance and without Poly-A tails. In addition, nuclei fraction enables separation of chromatin-associated scaffold RNAs from their cognate non-scaffold transcripts that have been released from chromatin.ConclusionsWe have developed a procedure for quantitative investigations on nucleic acids that are core components of RNA-directed DNA methylation. Our results not only demonstrated the efficacy of this procedure, but also provide lists of methylation-sensitive restriction enzymes, novel DNA methylation marker loci, and related siRNA sequences, all of which can be valuable for future epigenetic studies. Importantly, step-by-step protocols are provided in details such that the approaches can be easily followed by biologists with little experience in epigenetics.

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A Practical Guide to the Measurement and Analysis of DNA Methylation.

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