Development and Validation of Amplicon-Based Protocol for Sequencing of Respiratory Syncytial Virus Genome

Abstract

The most prevalent cause of severe respiratory infections in children is the human respiratory syncytial virus (RSV). The advent of next-generation sequencing (NGS) has made it possible to incorporate this technology into pathogen monitoring and surveillance. Whole-genome sequencing (WGS) of RSV has now become a relatively widely used method for tracking viral evolution. Here we report an improved high-throughput RSV-WGS assay performed directly on clinical samples that is suitable for short-read sequencing platforms. A total of 100 RSV-positive samples collected between November 2022 and March 2024 fulfilled the inclusion cycle quantification criteria and were randomly included in the validation process. The WGS protocol was designed to amplify three distinct amplicons to cover the entire RSV genome. The protocol described here can be successfully replicated in several instances (approximately 95%) in samples with a relatively low viral load, typically corresponding to cycle of quantification values of 27-32. The amplicon-based protocol produced meaningful sequencing results in terms of median depth of coverage (more than 12000×) and median of mapped reads (> 1 × 10⁶ reads). The sequences that had passed the filters showed a coverage of at least 98% across the entire genome, with cycle quantification values of 32. Based on the obtained data resulting in an easy-to-perform protocol helpful for the molecular epidemiology surveillance of RSV.

Keywords: amplicon‐based protocol; molecular epidemiology; next‐generation sequencing; respiratory syncytial virus; whole‐genome sequencing.

Figure 1

Phylogenetic and in silico analysis to assess the binding performance of primers for RSV‐A and RSV‐B. (A) The viral genome was divided into three segments with overlapping regions. Maximum likelihood phylogeny of RSV‐A (B) and RSV‐B (C) inferred from WGS. Phlyo‐primer mismatch graph to visualize of primer mismatches against the RSV‐A (D) and RSV‐B (E) phylogeny. Primer mismatches for each sequence were tabulated and overlaid on the tree. Sense primer, antisense primer, were evaluated in this order for each assay. Tip point colors represent the country of origin of RSV sequences including those originated from this study (in brown). Scale bars indicate 0.001 nucleotide substitution per site.

Figure 2

Genome coverage map for (A) RSV‐A and (B) RSV‐B. The mean coverage for each site is reported in violet and green with IQR; 25% and 75% in softened colour.

Figure 3

Violin plots showing (A) the mean depth (B) the percentage of RSV‐specific reads and (C) the mean mapped reads for RSV‐A in violet and RSV‐B in green. Correlation between Cq and (D) mean depth of coverage (E) the percentage of RSV‐specific reads and (F) mean of mapped reads for RSV‐A in violet and RSV‐B in green. Samples with Cq values ranging from 29 to 32 are indicated by a dot blue circle.