Self-Ligation-Free Next Generation Sequencing Adapters Applied to Methylation Assay

Abstract

In developing a bisulfite-free methylation assay with the use of restriction enzymes, self-ligation of next generation sequencing adapters (NGS-adapter) is a technological bottleneck to be overcome. In the experiments of this study, a variety of strategies designed to limit or abolish adapter’s self-ligation has been tested. Experimental data have showed that the three strategies tested can either substantially decrease or completely abolish the self-ligation of NGS-adapters. Minimization or elimination of NGS-adapter’s self-ligation is of importance in increasing the sensitivity, efficiency, and reproducibility of enzyme-mediated methylation assay. The strategies reported in the present study may find applications in some other nanotechnologies. In combination with nanotechnologies, either drop-digit PCR or microarray-based sequencing, the methylation-dependent endonuclease mediated methylation assays will facilitate applications of methylation analysis in both fundamental research and clinical epigenetic studies, particularly in early diagnosis of cancer.