An Improved Rapid and Sensitive Long Amplicon Method for Nanopore-Based RSV Whole-Genome Sequencing

Abstract

Background: Whole-genome sequencing (WGS) provides critical insights into the respiratory syncytial virus (RSV) transmission and any emerging mutations that could impair the efficacy of monoclonal antibodies or vaccines that have been recently licenced for clinical use worldwide. However, the ability to sequence RSV genomes at large scale is limited by expensive and time-consuming sequencing methods. Oxford Nanopore Technology (ONT) offers significant improvements in next generation sequencing (NGS) both in turnaround time and cost, compared with other platforms for viral WGS.

Methods: We have developed and modified an RSV long amplicon-based WGS protocol for the ONT platform using a one-step multiplex RT-PCR assay and the rapid barcoding kit. One hundred thirty-five RSV positive Australian clinical specimens (91 RSV-A and 44 RSV-B) sampled in 2023 with cycle threshold (Ct) values between 14 to 35 were tested in this study. This ONT workflow was compared with other recent RSV WGS amplification assays based on short amplicons.

Results: A PCR amplicon clean-up step prior to library preparation significantly improved WGS result for samples with poor amplicon generation, but it is not necessary or beneficial for ones that generated high concentrations of amplicons. Overall, a success rate of 85.9% was achieved for WGS. This method performed as well as the more complex short amplicon methods in terms of genome coverage and sequencing depth.

Conclusions: The workflow described here was highly successful in generating RSV WGS on ONT platform and had improved turnaround times and excellent results with RSV clinical samples with Ct values up to 30.

Keywords: ONT; rapid barcoding; respiratory syncytial virus; whole‐genome sequencing.

FIGURE 1

The performance of ONT rapid barcoding method mediated NGS with library preparation from samples with sufficient amplicons. (A) Histograms of the Ct value distribution for clinical samples having sufficient (dark green) and insufficient (dark red) amplicons generated for ONT libraries. (B) Histograms of the Ct value distribution for clinical samples within sufficient amplicons group with RSV full‐length genome (blue), G plus F genes (brown), only G gene (dark red) and missing G and F gene sequenced (also called ‘other’), respectively. (C) Coverage depth of sequenced representative RSV‐A (red) and RSV‐B (blue) in genomic position covered by six overlapping amplicons. (D) Histograms of the Ct value distribution for clinical samples within sufficient amplicons group having RSV whole genome (blue), G plus F genes (brown), only G gene (dark red) and missing G and F gene sequenced (also called ‘other’ in dark green), whole genome rescued by DNA purification (purple), respectively. Coverage depth of sequenced representative RSV‐A (E) and RSV‐B (F) in genomic position with (blue) and without (red) PCR amplicon clean‐up. Bar plots showing the fold change of NGS reads mapped to RSV reference genomes from sequenced samples of low (G) and high (H) concentration of amplicons generated without and with PCR amplicon purification. NGS reads of libraries from washed PCR amplicons were normalised and expressed as fold changes to ones from unwashed PCR amplicons that were set to 1. Data are expressed as mean ± SD. t‐test analysis was performed for statistical significance. P values less than 0.05 were considered as statistically significant and labelled as * in the figures.

FIGURE 2

RSV NGS achieved by one long and two short amplicon‐based RSV WGS assays. Coverage depth of sequenced representative RSV‐A (A) and RSV‐B (B) in genomic position with long amplicon‐based (red), Maloney's short amplicon‐based (green) and Talts' short amplicon‐based (blue) assays.

FIGURE 3

Phylogenetic analysis of full‐length of RSV‐A and RSV‐B. (A) Phylogenetic analysis of full‐length of RSV‐A generated using the ONT workflow described in this method. RSV‐A full‐length genome sequences generated using this method were analyzed phylogenetically and showed that the RSV‐A genomes clustered into 3 main clades. (B) Phylogenetic analysis of full‐length of RSV‐B generated using the ONT workflow described in this method. RSV‐B full‐length genome sequences generated using this method were analysed phylogenetically and showed that the RSV‐B genomes clustered into two main clades.

FIGURE 4

Schematic diagram of ONT NGS workflow for RSV WGS based on a long PCR amplicon assay.