The Oxford Nanopore MinION: delivery of nanopore sequencing to the genomics community

Abstract

Nanopore DNA strand sequencing has emerged as a competitive, portable technology. Reads exceeding 150 kilobases have been achieved, as have in-field detection and analysis of clinical pathogens. We summarize key technical features of the Oxford Nanopore MinION, the dominant platform currently available. We then discuss pioneering applications executed by the genomics community.

Fig. 1

Data for a 2D read of a full-length λ phage dsDNA from the MinION nanopore sequencer. a Steps in DNA translocation through the nanopore: (i) open channel; (ii) dsDNA with lead adaptor (blue), bound molecular motor (orange) and hairpin adaptor (red) is captured by the nanopore; capture is followed by translocation of the (iii) lead adaptor, (iv) template strand (gold), (v) hairpin adaptor, (vi) complement strand (dark blue) and (vii) trailing adaptor (brown); and (viii) status returns to open channel. b Raw current trace for the passage of the single 48-kb λ dsDNA construct through the nanopore. Regions of the trace corresponding to steps i–viii are labeled. (c) Expanded time and current scale for raw current traces corresponding to steps i–viii. Each adaptor generates a unique current signal used to aid base calling

Fig. 2

‘Read Until’ strategy for selective sequencing of dsDNA molecules. The ionic current profile obtained during translocation of a DNA strand through the nanopore is compared in real time to the ionic current profile of a target sequence. a As sequencing of the template strand of DNA proceeds (during step iv), the measured current is compared to the reference current profile. If there is a match, sequencing of that strand continues to completion (steps v–vii). A new strand can now be captured. b Alternatively, if the measured current does not match the reference current profile, the membrane potential is reversed, sequencing of that strand stops, and the strand is ejected (at stage v). A new strand can now be captured. (Image based on the strategy of Loose et al. [43])

Fig. 3

Estimate CT47-repeat copy-number on human chromosome Xq24. a BAC end sequence alignments (RP11-482A22: AQ630638 and AZ517599) span a 247-kb region, including 13 annotated CT47 genes [69] (each within a 4.8-kb tandem repeat), and a 50-kb scaffold gap in the GRCh38/hg38 reference assembly. b Nine MinION reads from high molecular weight BAC DNA span the length of the CT47-repeat region, providing evidence for eight tandem copies of the repeat. The insert (dashed line), whose size is estimated from pulse-field gel electrophoresis, with flanking regions (black lines) and repeat region (blue line) are shown. Single-copy regions before and after the repeats are shown in orange (6.6 kb) and green (2.6 kb), respectively, along with repeat copies (blue) and read alignment in flanking regions (gray). The size of each read is shown to its left. c Shearing BAC DNA to increase sequence coverage provided copy-number estimates by read depth. All bases not included in the CT47 repeat unit are labeled as flanking regions (gray distribution; mean of 46.2-base coverage). Base coverage across the CT47 repeats was summarized over one copy of the repeat to provide an estimate of the combined number (dark blue distribution; mean of 329.3-base coverage) and was similar to single-copy estimates when normalized for eight copies (light blue distribution; mean of 41.15-base coverage). (Figure reproduced from Jain et al. [9])

Fig. 4

Maximum-likelihood alignment parameters derived using expectation-maximization (EM). The process starts with four guide alignments, each generated with a different mapper using tuned parameters. Squares denote error estimates derived from different mappers when used without tuning; circles denote error estimates post-tuning; and triangles denote error estimates post-EM. a Insertion versus deletion rates, expressed as events per aligned base. b Indel events per aligned base versus rate of mismatch per aligned base. Rates varied strongly between different guide alignments; but EM training and realignment resulted in very similar rates (gray shading in circles), regardless of the initial guide alignment. c The matrix for substitution emissions determined using EM reveals very low rates of A-to-T and T-to-A substitutions. The color scheme is fitted on a log scale, and the substitution values are on an absolute scale. (Figure reproduced from Jain et al. [9])