An Amplicon-Based Approach for the Whole-Genome Sequencing of Human Metapneumovirus

Abstract

Human metapneumovirus (HMPV) is an important cause of upper and lower respiratory tract disease in individuals of all ages. It is estimated that most individuals will be infected by HMPV by the age of five years old. Despite this burden of disease, there remain caveats in our knowledge of global genetic diversity due to a lack of HMPV sequencing, particularly at the whole-genome scale. The purpose of this study was to create a simple and robust approach for HMPV whole-genome sequencing to be used for genomic epidemiological studies. To design our assay, all available HMPV full-length genome sequences were downloaded from the National Center for Biotechnology Information (NCBI) GenBank database and used to design four primer sets to amplify long, overlapping amplicons spanning the viral genome and, importantly, specific to all known HMPV subtypes. These amplicons were then pooled and sequenced on an Illumina iSeq 100 (Illumina, San Diego, CA, USA); however, the approach is suitable to other common sequencing platforms. We demonstrate the utility of this method using a representative subset of clinical samples and examine these sequences using a phylogenetic approach. Here we present an amplicon-based method for the whole-genome sequencing of HMPV from clinical extracts that can be used to better inform genomic studies of HMPV epidemiology and evolution.

An Unusual Resurgence of Human Metapneumovirus in Western Australia Following the Reduction of Non-Pharmaceutical Interventions to Prevent SARS-CoV-2 Transmission

“…A purposively sampled selection of hMPV-positive routine diagnostic specimens was chosen ensuring geographical and temporal representation from 2017 to 2021. Nucleic acid was extracted, and tiled amplicon sequencing was performed using established protocols [ 22 , 23 ]. In brief, four primer sets were used to amplify overlapping amplicons spanning the hMPV genome.…”

Introducing clinical nanoarchaeaology: Isolation by co-culture of Nanopusillus massiliensis sp. nov.

“…Finishing was achieved by LongRange PCR (LR-PCR) followed by next-generation sequencing after PCR primers were designed by aligning nanoarchaea on-going genome sequence with Candidatus N. acidilobi reference genome, targeting regions to be finished (SI Appendix, Table S5) . Purified LR-PCR products were sequenced using the Illumina Iseq technique as previously described ( Tulloch et al., 2021 ). Contigs were identified using Blastn against NCBI database and concatenated reads were mapped using CLCgenomic version 7 against the Candidatus N. acidilobi genome sequence.…”

Prophylactic Ribonucleic Acid Vaccines to Combat RNA Viral Infections in Humans

“…HMPV became a part of infectious disease genomic surveillance after development of whole-genome tiled amplicon sequencing technology. This methodology allowed the identification of two major phylogenetic subtypes of HMPV, each containing two sublineages (A1, A2, B1, B2) [83,84]. The use of this new knowledge in vaccine manufacturing led to multi-viral vaccine research and development.…”