Nanopore Sequencing of Short dsDNA after Elongation by Combination of Ligation and PEAR

Abstract

Abstract Nanopore sequencing technology, as a third-generation method for DNA sequencing at the single-molecule level, has attracted much attention and developed quickly due to the advantages of low cost and ultra-long read. However, nanopore sequencing of short DNAs (<500 bp) is not suitable due to the unfavorable cost performance and complicated data analysis. Here, we describe a novel method for nanopore sequencing using short dsDNA elongation by ligation and PEAR (SELP). Before sequencing, short PCR products are subjected to intermolecular ligation and subsequent elongation using PEAR (Polymerase-Endonuclease Amplification Reaction). The obtained long concatemers (thousands of base pairs) of repetitive DNA sequences are ideal samples for nanopore sequencing. The sequencing results demonstrate that short dsDNA can be elongated by more than 20 fold, and precise sequence analysis can be obtained through a single read. Accordingly, SELP-Seq can be used for simultaneous sequencing of multiple (even thousands of) short dsDNAs. Obviously, our approach can greatly expand the applications of nanopore sequencing, such as SNP analysis and high-throughput DNA detection.