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

Area of Science:

• Genomics
• Epigenetics
• Molecular Biology

Background:

• DNA methylation is a key regulator of gene expression.
• Current methods for targeted genome analysis are limited to large regions.
• Studying specific methylation patterns is crucial for understanding gene regulation.

Purpose of the Study:

• To develop a protocol for epigenotyping differentially methylated CpGs in targeted genomic regions.
• To provide a high-throughput, cost-effective alternative to existing methylation analysis technologies.
• To enable custom panel adaptation for various species.

Main Methods:

• A targeted methylation library preparation protocol using enzymatic conversion (New England Biolabs Next Enzymatic Methyl-seq) and Twist Bioscience Targeted Methylation Sequencing.
• The protocol involves fragmentation, library preparation, enzymatic conversion, indexing, pooling, hybridization, capture, and amplification.
• Short-read sequencing on an Illumina instrument.

Main Results:

• The protocol requires low DNA input (50-100 ng).
• Enzymatic conversion minimizes DNA degradation compared to chemical bisulfite methods.
• 8-plex pooling reduces handling time, and panel quantity reduction saves costs.
• The method was validated for simultaneous analysis of 96 samples, with scalability up to 384 samples.

Conclusions:

• A novel, high-throughput epigenotyping method for targeted DNA methylation analysis has been developed.
• This protocol offers advantages in DNA input, reduced degradation, efficiency, and cost-effectiveness.
• The adaptable approach serves as a valuable tool for epigenomic research in agronomic and model organisms.