Nanopore Sequencing as a Rapidly Deployable Ebola Outbreak Tool

Abstract

Rapid sequencing of RNA/DNA from pathogen samples obtained during disease outbreaks provides critical scientific and public health information. However, challenges exist for exporting samples to laboratories or establishing conventional sequencers in remote outbreak regions. We successfully used a novel, pocket-sized nanopore sequencer at a field diagnostic laboratory in Liberia during the current Ebola virus outbreak.

Keywords: DNA; Ebola hemorrhagic fever; Ebola virus; Ebola virus disease; Ebolavirus; Liberia; West Africa; disease outbreaks; high-throughput nucleotide sequencing; molecular diagnostic techniques; nanopore sequencing; viruses.

Figure 1

MinION sequencing. A) Experimental and B) bioinformatics workflows. Times indicated are the approximate duration for each procedure. RT, reverse transcription. C) Sequencing results showing Ebola virus load (expressed as C_t value), percentage of the genome with a minimum read depth of >1 or >33, mean read depth, theoretical probability for a miscalled base (TPMB), and GenBank accession numbers of complete and nearly complete genomes. Brackets at left indicate percentage of Ebola virus-positive patient samples below each of the 3 cutoff cycle threshold (C_t) values used in this study (C_t <21, <24, <31). Sample 8 was from an oral swab; all others were from blood. NA, not available.

Figure 2

Observed mutations in the 8 fully nanopore-sequenced Ebola-positive blood samples compared to a reference sequence from June 2014 (SLI/Makona-EM106, GenBank accession number KM233036.1). Squares indicate nonsynonymous mutations, circles indicate synonymous changes, and triangles indicate changes in noncoding regions.