Multiplexed Targeted Sequencing for Oxford Nanopore MinION: A Detailed Library Preparation Procedure

Abstract

MinION is a small form factor sequencer recently retailed by Oxford Nanopore technologies. This lighter-sized USB3.0-interfaced device uses innovative nanotechnology to generate extra-long reads from libraries prepared using only standard molecular biology lab equipment. The flexibility and the portability of the platform makes it ideal for point-of-interest and real-time surveillance applications. However, MinION's limited capacity is not enough for the study of specific targets within larger genomes. Apart from just PCR-amplifying regions of interest, the capture of long reads spanning the edges of known-unknown genomic regions is of great importance for structural studies, such as the identification of mobile elements' integrations sites, bridging over low complexity repetitive regions etc.In this study, using MinION-kit-included and commercially available reagents, we have developed an easy and versatile wet-lab procedure for the targeted enrichment of MinION libraries, capturing DNA fragments of interest before the ligation of the sensitive MinION sequencing-adapters. This method allows for simultaneous target-enrichment and barcode-multiplexing of up to 12 libraries, which can be loaded in the same sequencing run.

Keywords: Baits; Hybridization; Library preparation; MinION; Target-enrichment.