Assessing the performance of the Oxford Nanopore Technologies MinION

Abstract

The Oxford Nanopore Technologies (ONT) MinION is a new sequencing technology that potentially offers read lengths of tens of kilobases (kb) limited only by the length of DNA molecules presented to it. The device has a low capital cost, is by far the most portable DNA sequencer available, and can produce data in real-time. It has numerous prospective applications including improving genome sequence assemblies and resolution of repeat-rich regions. Before such a technology is widely adopted, it is important to assess its performance and limitations in respect of throughput and accuracy. In this study we assessed the performance of the MinION by re-sequencing three bacterial genomes, with very different nucleotide compositions ranging from 28.6% to 70.7%; the high G + C strain was underrepresented in the sequencing reads. We estimate the error rate of the MinION (after base calling) to be 38.2%. Mean and median read lengths were 2 kb and 1 kb respectively, while the longest single read was 98 kb. The whole length of a 5 kb rRNA operon was covered by a single read. As the first nanopore-based single molecule sequencer available to researchers, the MinION is an exciting prospect; however, the current error rate limits its ability to compete with existing sequencing technologies, though we do show that MinION sequence reads can enhance contiguity of de novo assembly when used in conjunction with Illumina MiSeq data.

Keywords: DNA sequencing; MinION; NRPS, non-ribosomal peptide synthase; Nanopore; ONT, Oxford Nanopore Technologies.

Fig. 1

Distribution of MinION read lengths. Frequency distributions of lengths of reads obtained from the MinION run. Data shown for each of the three read types Template, Complement and Two direction, superimposed.

Fig. 2

G + C content of MinION reads. A frequency distribution of the G + C content of reads generated by the MinION run. Mean G + C content of each reference genome is included for comparison.

Fig. 3

G + C content of aligned portions of MinION reads against corresponding reference sequence. Plot of G + C content of the aligned portion of a read versus the G + C content of the section of the reference to which it aligns. Included is a line to demonstrate the relationship if the two were equal.

Fig. 4

G + C content of aligned portions of MinION reads and the reference sequence aligned to. Frequency distribution of the G + C content of aligned portions of the reads (A) and the G + C content of the sections of the reference genome they align to (B). Mean G + C content of each reference genome is included for comparison.

Fig. 5

Single MinION reads able to span important genes. Images generated using IGV showing the alignment of MinION reads to the E. coli reference genome. (A) A rDNA operon. (B) A NRPS gene cluster. Highlighted with continuous red lines are the reads spanning the relevant sections and the dashed lines highlight the genomic regions of interest.

Fig. 6

Read data over time during the MinION run. Plot of number of reads and their mean length generated per hour during the MinION. Additional input material was added to the MinION flowcell at 16 h 33 min.

Fig. 7

Fluctuation in alignment and error rates over time during a MinION run. Plot showing percentage of read bases aligned per hour during the MinION run based on the alignment by LAST and their error rate. Additional input material was added to the MinION flowcell at 16 h 33 min.