An Evaluation of Avian Influenza Virus Whole-Genome Sequencing Approaches Using Nanopore Technology

Abstract

As exemplified by the global response to the SARS-CoV-2 pandemic, whole-genome sequencing played an important role in monitoring the evolution of novel viral variants and provided guidance on potential antiviral treatments. The recent rapid and extensive introduction and spread of highly pathogenic avian influenza virus in Europe, North America, and elsewhere raises the need for similarly rapid sequencing to aid in appropriate response and mitigation activities. To facilitate this objective, we investigate a next-generation sequencing platform that uses a portable nanopore sequencing device to generate and present data in real time. This platform offers the potential to extend in-house sequencing capacities to laboratories that may otherwise lack resources to adopt sequencing technologies requiring large benchtop instruments. We evaluate this platform for routine use in a diagnostic laboratory. In this study, we evaluate different primer sets for the whole genome amplification of influenza A virus and evaluate five different library preparation approaches for sequencing on the nanopore platform using the MinION flow cell. A limited amplification procedure and a rapid procedure are found to be best among the approaches taken.

Keywords: avian influenza; nanopore sequencing; next-generation sequencing; whole-genome sequencing.

Figure 1

Net cDNA generated with four primer sets. The amount, in nanograms, of cDNA recovered following bead purification of the SuperScript III RT-PCR reaction on the three HPAI H5N1 swab samples is shown on the Y axis. Primer sets used (1–4, see Table 2) are listed on the X axis and the RNA samples used (from Table 1) are listed in the figure legend.

Figure 2

Treemap representation of the time required for the methods evaluated.