Human Respiratory Syncytial Virus (RSV) belongs to the Pneumoviridae family and is a common cause of severe respiratory tract infections in infants, young children, older adults and immunocompromised individuals [1,2]. Despite the significant public health impact and global economic burden of RSV infections [3,4], there are no vaccines or effective treatments currently available. The only prevention currently is an expensive monoclonal antibody prophylaxis for young children [5], [6], [7].

RSV is divided into two major types RSV-A and RSV-B [8]. Most genetic studies of RSV are focused on the attachment glycoprotein (G) gene, which is the most variable region and has been commonly used for RSV genotyping [9]. In contrast, the F protein (which is responsible for fusion of viral and host cell membranes and syncytium formation) is highly conserved between strains and is the target of most vaccine and monoclonal antibody treatments currently being developed [10]. The mutations acquired during evolution may lead to changes in various aspects of virus biology, such as infectivity, antigenicity, drug resistance and potentially virulence [11,12]. Therefore, there is an ongoing need for effective monitoring of RSV viral evolution and spread throughout human society.

Among different Next generation sequencing (NGS) approaches, the PCR amplicon-based NGS is a sensitive method for sequencing small genomes such as RSV for population-scale viral surveillance [13]. One challenge associated with this method is the generation of PCR amplicons that can cover all variants. Several singleplex reverse‐transcription (RT)-PCR amplicon-based NGS methods have been used for RSV whole-genome sequencing (WGS) including a commonly used method that requires at least four separate reactions per genome [14,15]. While the use of multiplex PCR (mPCR) to amplify multiple DNA fragments in the same reaction provides a simpler solution to reducing the number of reactions [16], it often leads to a decreased sensitivity, due to complex primer-primer interactions and competition for usage of reagents [17]. Therefore, the establishment of optimal primer pools for mPCR can be challenging and may require many sets of primers as seen with the ARTIC protocol for COVID-19 WGS [18,19]. The development of amplicon-based WGS should ideally be suitable for multiple NGS platforms, especially in developing countries where the burden of RSV is greatest. One such system that is currently widely used is the ONT's MinION system, which is a portable and low-cost NGS that can significantly simplify the sequencing workflow and shorten the turnaround time for sequencing [20].

In April 2019, the World Health Organization (WHO) initiated phase 2 of their Global RSV Surveillance program to study the circulation and impact of RSV worldwide. One of the elements of this study was to increase the sequencing capacity for RSV viruses [21], especially in developing countries. In order to meet the increasing demand for RSV sequencing, we endeavoured to develop a simplified one-step multiplex RT-PCR (mRT-PCR) method for RSV WGS.