Amplicon-Based Bisulfite Conversion-NGS DNA Methylation Analysis Protocol

DNA methylation is an important epigenetic modification that regulates chromatin structure and the cell-type-specific expression of genes. The association of aberrant DNA methylation with many diseases, as well as the increasing interest in modifying the methylation mark in a directed manner at genomic sites using epigenome editing for research and therapeutic purposes, increases the need for easy and efficient DNA methylation analysis methods. The standard approach to analyze DNA methylation with a single-cytosine resolution is bisulfite conversion of DNA followed by next-generation sequencing (NGS). In this chapter, we describe a robust, powerful, and cost-efficient protocol for the amplification of target regions from bisulfite-converted DNA, followed by a second PCR step to generate libraries for Illumina NGS. In the two consecutive PCR steps, first, barcodes are added to individual amplicons, and in the second PCR, indices and Illumina adapters are added to the samples. Finally, we describe a detailed bioinformatics approach to extract DNA methylation levels of the target regions from the sequencing data. Combining barcodes with indices enables a high level of multiplexing allowing to sequence multiple pooled samples in the same sequencing run. Therefore, this method is a robust, accurate, quantitative, and cheap approach for the readout of DNA methylation patterns at defined genomic regions.