Direct bisulfite sequencing for examination of DNA methylation with gene and nucleotide resolution from brain tissues

Abstract

DNA methylation is an epigenetic modification that is essential for the development and mature function of the central nervous system. Due to the relevance of this modification to the transcriptional control of gene expression, it is often necessary to examine changes in DNA methylation patterns with both gene and single-nucleotide resolution. Here, we describe an in-depth basic protocol for direct bisulfite sequencing of DNA isolated from brain tissue, which will permit direct assessment of methylation status at individual genes as well as individual cytosine molecules/nucleotides within a genomic region. This method yields analysis of DNA methylation patterns that is robust, accurate, and reproducible, thereby allowing insights into the role of alterations in DNA methylation in brain tissue.

Figure 1

Schematic diagram of direct bisulfite sequencing workflow. DNA is first bisulfite treated to conserve methylated cytosine while unmethylated cytosine is converted to uracil. During PCR, uracil is converted to thymine and product is amplified. PCR product is then sequenced. The methylation level of each CpG in the amplicon will result in differing peak sizes for cytosine and thymine as read on the chromatogram.

Figure 2

An example of how to perform data analysis of a single CpG site with use of the forward primer for sequencing. Chromatogram peaks for thymine (representing unmethylated cytosines that were converted to uracil during bisulfite treatment) and cytosine (representing methylated cytosines that were spared during bisulfite treatment) are compared to determine the average level of methylation for each site within a given sample. The same analysis should be applied when using the reverse primers for sequencing except methylation levels will be derived using the ratio of adenine (unmethylated) to guanine (methylated) instead of thymine and cytosine.

Figure 3

Verification of bisulfite sequencing using DNA methylation standards. A) Schematic of a region of the Arc gene with known CpG sites (tick marks), and CpG island (gray box). The 7 CpG sites enlarged are the sites used to determine efficiency of direct bisulfite sequencing at the Arc gene. B) Accuracy of direct bisulfite sequencing. Graph shows actual average methylation versus expected average methylation of known quantities of methylated to unmethylated DNA (using EpigenDx premixed DNA standards). As outlined in the text, bisulfite sequencing resulted in DNA methylation levels that bore a significant linear relationship to expected methylation levels. C) Actual CpG methylation for each site in the amplified region from A, shown for each level of expected methylation (0–100%). Results indicate accuracy of direct bisulfite sequencing across sites. D) Reliability of direct bisulfite sequencing. Independent DNA standard samples were processed for bisulfite sequencing. Results indicate a highly significant and robust linear relationship between two independent replicates – an original (abscissa) and a replicate (ordinate).