Construction of Non-Self-Ligating Blunt-End Adapters for Sequencing by Leveraging the Asymmetry of T4 DNA Ligase

Efficient and specific adapter ligation is essential for high-quality library construction in high-throughput sequencing. Conventional blunt-end adapters offer superior ligation efficiency compared to 3'-T overhang adapters but suffer from severe self-ligation, resulting in unwanted adapter dimers and reduced library purity. Here, we report a new class of Non-Self-Ligating Blunt-End (NSL-BE) adapters enabled by the intrinsically asymmetric DNA-binding requirements of T4 DNA ligase, which imposes distinct minimal duplex-length constraints at the 3'-hydroxyl and 5'-phosphate sides of a ligation junction. By rationally designing blunt-end adapters consisting of a 5-bp duplex (serving as the blunt-end) and adjacent mismatches, self-ligation is effectively suppressed, while efficient ligation to target fragments is retained. Under standard T4 DNA ligase buffer conditions, NSL-BE adapters achieve >85% paired-end ligation efficiency, substantially outperforming T-adapters. Importantly, NSL-BE adapters incorporating only a minimal ligation module were successfully applied to high-throughput sequencing (Illumina) of genomic DNA, demonstrating their practical sequencability and compatibility with real library construction workflows. Modular extension of the adapter structure allows the incorporation of sequencing elements, functional domains, and nanostructure interfaces, supporting adaptation across diverse library formats and sequencing platforms. Beyond blunt-end ligation, this strategy has been successfully extended to sticky-end adapters with palindromic overhangs, demonstrating its broader utility. Altogether, this work presents a facile and powerful solution for eliminating adapter self-ligation with broad applications in high-throughput sequencing, biotechnology, and enzymology.