Oxford Nanopore MinION Sequencing and Genome Assembly

Abstract

The revolution of genome sequencing is continuing after the successful second-generation sequencing (SGS) technology. The third-generation sequencing (TGS) technology, led by Pacific Biosciences (PacBio), is progressing rapidly, moving from a technology once only capable of providing data for small genome analysis, or for performing targeted screening, to one that promises high quality de novo assembly and structural variation detection for human-sized genomes. In 2014, the MinION, the first commercial sequencer using nanopore technology, was released by Oxford Nanopore Technologies (ONT). MinION identifies DNA bases by measuring the changes in electrical conductivity generated as DNA strands pass through a biological pore. Its portability, affordability, and speed in data production makes it suitable for real-time applications, the release of the long read sequencer MinION has thus generated much excitement and interest in the genomics community. While de novo genome assemblies can be cheaply produced from SGS data, assembly continuity is often relatively poor, due to the limited ability of short reads to handle long repeats. Assembly quality can be greatly improved by using TGS long reads, since repetitive regions can be easily expanded into using longer sequencing lengths, despite having higher error rates at the base level. The potential of nanopore sequencing has been demonstrated by various studies in genome surveillance at locations where rapid and reliable sequencing is needed, but where resources are limited.

Keywords: De novo assembly; Molecular clinical diagnostics; Oxford nanopore MinION device; Structural variations; Third-generation sequencing.

Figure 1

The MinION sequencing device DNA sequencing is performed by adding the sample to the flowcell. When DNA molecules pass through or near the nanopore, there will be a change in the magnitude of the current in the nanopore, which is measured by a sensor. The data streams are passed to the ASIC and MinKNOW, the software that generates the signal-level data. ASIC, application-specific integrated circuit.

Figure 2

Flow of Oxford Nanopore sequencing process A. Library preparation schematic for the genomic DNA sequencing kit (SQK-MAP-003). B. 512 channels with different levels of activity in a flowcell are shown in different color (most active channels are in red). C. “Squiggle plots” of fluctuating electrical signals, which can be translated into DNA bases. D. 5-mers decoding from event information and alignment of 1D and 2D base calls.

Figure 3

Time course of base throughput and read length distribution A. The throughput of bases with time generated using Poretools . B. Distribution of read length using data from one flowcell run .

Figure 4

Counts of 5-mers from the reference and the assembly before and after polishing using event data A. Counts of 5-mer homopolymers before event polishing. B. Counts of 5-mer homopolymers after Nanopolish using event data.