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

Nathalie Iannuccelli1, Sophie Valière2, Julien Sarry1

  • 1GenPhySE, Université de Toulouse, INRAE, ENVT, Castanet-Tolosan, France.

FEBS Open Bio
|August 14, 2025
PubMed
Summary

We developed a new protocol for targeted DNA methylation analysis, enabling precise epigenotyping of specific genomic regions. This method offers a cost-effective, high-throughput alternative to current technologies for gene expression studies.

Keywords:
DNA methylationbiotechnologycustom captureenzymatic conversionhybridizationmolecular biology

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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.