Revisiting Sequencing by Hybridization at the Single Molecule Level using the Unzipping Assay

Improving NGS is the major challenge of the third-generation sequencing which is mainly focus on single molecule techniques. Single molecule offers the ultimate sensitivity, avoiding the amplification steps which introduces bias and strongly impairs epigenetic analysis. However, single molecule approaches requires minute signal detection which results in an increased error rate. We present here a third method where a single molecule can be interrogated in a repeated manner allowing good signal averaging and leading to very low error rate. This method may be seen as revisiting Sequencing By Hybridization (SBH) using a hairpin re-zipping blocking assay. In our single molecule micromanipulation assay we detect the hybridization of the oligonucleotides k-mers and their actual position along the target with an accuracy of a few bases. Each repeat is thus identified by its actual position. This process is repeated until all hybridization positions are known accurately. Assembling the target sequence is achieved after testing a series of oligonucleotides once the target sequence is sufficiently covered. The overlapping of the k-mers is used by a software to reconstruct the sequence.