An optimized protocol to detect high-throughput DNA methylation from custom targeted sequences on 96 samples simultaneously.

Genome methylation represents an important source of regulation of gene expression. To date, custom molecular tools for studying targeted regions of the genome are restricted to several megabases. We developed a protocol to epigenotype differentially methylated CpGs in specific regions of the genome. The protocol describes a targeted methylation library preparation upstream short read sequencing with an Illumina instrument. The protocol includes the New England Biolabs Next Enzymatic Methyl-seq Library Preparation workflow combined with the Twist Bioscience Targeted Methylation Sequencing workflow. The protocol is divided into eight steps: fragmentation, library preparation, enzymatic conversion, indexing, pooling, hybridization, capture, and amplification. Main advantages are (a) a lower amount of DNA (100 and 50 ng) than other technologies, (b) the limitation of DNA degradation using enzymatic conversion instead of chemical bisulfite, (c) the pooling of samples into 8-plex reducing handling time, and (d) the significant reduction of the panel quantity divided by 20 for saving experimental costs. This protocol was carried out on 96 samples simultaneously in a standard molecular biology laboratory, and the multiplexing can be run up to 384 samples for methylation experiments. We developed a high-throughput epigenotyping method as an alternative of methylation arrays. This approach can be adapted to any interesting regions using a custom panel for agronomic species and model organisms.