Nickase-based suppression of self-ligation in long sticky-end adapters for next-generation sequencing library preparation

The correctness and efficiency of the adapter connection are important factors affecting the quality and yield of the libraries in next-generation sequencing (NGS). Lengthening the sticky ends of the adapters and the targets can effectively improve the efficiency of ligation, however, it may cause a new trouble of adapter self-ligation. In this study, a strategy based on nickase is designed to limit or abolish long sticky-end adapters self-ligation. We firstly explored the enzymatic characteristics of Nt.BsmAI, a typical nickase. Then based on the working properties of Nt.BsmAI to design Y-adapters for NGS and evaluated the efficiency of this strategy for reducing long sticky-end adapter dimers. Moreover, the feasibility of Nb.BtsI (another nickase) was verified using the same strategy. This study provides new application for nickase in NGS library construction, and the possibility of using multi-base sticky end adapters in the future. NGS adapters with long sticky-ends paves a new avenue for enhancing the application performance of NGS, particularly for methylation sequencing, in biomedicine, with a higher efficiency in library preparation.