MS-HRM protocol: a simple and low-cost approach for technical validation of next-generation methylation sequencing data

Mol Genet Genomics. 2022 Jul;297(4):1101-1109. doi: 10.1007/s00438-022-01906-1. Epub 2022 May 26.

Abstract

DNA methylation is a fundamental epigenetic process and have a critical role in many biological processes. The study of DNA methylation at a large scale of genomic levels is widely conducted by several techniques that are next-generation sequencing (NGS)-based methods. Methylome data revealed by DNA methylation next-generation sequencing (mNGS), should be always verified by another technique which they usually have a high cost. In this study, we offered a low-cost approach to corroborate the mNGS data. In this regard, mNGS was performed on 6 colorectal cancer (case group) and 6 healthy individual colon tissue (control group) samples. An R-script detected differentially methylated regions (DMRs), was further validated by high resolution melting (MS-HRM) analysis. After analyzing the data, the algorithm found 194 DMRs. Two locations with the highest level of methylation difference were verified by MS-HRM, which their results were in accordance with the mNGS. Therefore, in the present study, we suggested MS-HRM as a simple, accurate and low-cost method, useful for confirming methylation sequencing results.

Keywords: Bisulfite; DNA Methylation Sequencing; High resolution melting; Validation.

MeSH terms

  • DNA Methylation* / genetics
  • Genomics
  • High-Throughput Nucleotide Sequencing*
  • Polymerase Chain Reaction / methods
  • Sequence Analysis, DNA / methods