New approaches for adapter synthesis in Duplex Sequencing
Sprache des Vortragstitels:
European Human Genetics Conference 2019
Sprache des Tagungstitel:
Introduction: Duplex Sequencing (DS) is a next generation sequencing methodology capable of detecting ultra-low mutation frequencies by exploiting the fact that genetic information is stored on complementary DNA strands. Therefore, DNA fragments are ligated to adapters containing a double stranded degenerate sequence. Currently, fork adapters consisting of two partially complementary oligonucleotides are being used in DS. However, it has been suggested that adapters comprising a hairpin loop structure, such as the NEBNext adapters, show improved performance in library preparation and sequencing. Here, we propose different synthesis methods for creating hairpin loop adapters for DS in house.Material and Methods: We have developed a synthesis strategy for a hairpin adapter and for adapters containing a phosphorothioate bond at the ligation site. Currently we use an adapted protocol for DS library preparation with those loop adapters. Adapter synthesis and proper function has been verified by gel electrophoresis and sequencing.Results: We have successfully synthesized the proposed hairpin adapters and replaced the fork adapter used in the original protocol by Schmitt et al. (2012) with our loop adapters. Sequencing data confirm their experimental feasibility.Discussion: Our hairpin adapters might offer advantages over previously used fork adapters due to structural differences. Additionally, we integrated a phosphorothioate bond into DS hairpin adapters which might increase the stability of the ligation site, reduce removal of the 3? T-overhang and thus, decrease potential adapter dimer formation between blunt-ended sites.