An optimized protocol for high-throughput custom targeted sequence DNA methylation enrichment

This protocol describes a methylated targeted library preparation for Illumina DNA sequencing. The protocol is an in-house combination of 2 protocols : NEBNext Enzymatic Methyl-seq Library Preparation Protocol and TwistBioscience Targeted Methylation Sequencing Protocol. The workflow is divided into 8 steps including an enzymatic conversion followed by a custom capture. Then, DNA degradation is avoided and off target is reduced. The workflow takes 3 days working with several break points. The critical steps are hybridization/capture due to the temperature control to ensure probes specificity. Samples were pooled into 8-plex to reduce handling time and capture costs. This protocol can be performed in standard molecular biology laboratories and it's suitable for 96 samples. The use of a methyl custom panel allows to target many and very GC rich regions with different sizes. This approach can be adapted to any interest regions of any agronomic species and could even be used as an human diagnostic tool.