Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment

Abstract

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract infections in children worldwide, while human noroviruses (HuNoV) are a leading cause of epidemic and sporadic acute gastroenteritis. Generating full-length genome sequences for these viruses is crucial for understanding viral diversity and tracking emerging variants. However, obtaining high-quality sequencing data is often challenging due to viral strain variability, quality, and low titers. Here, we present a set of comprehensive oligonucleotide probe sets designed from 1,570 RSV and 1,376 HuNoV isolate sequences in GenBank. Using these probe sets and a capture enrichment sequencing workflow, 85 RSV positive nasal swab samples and 55 (49 stool and six human intestinal enteroids) HuNoV positive samples encompassing major subtypes and genotypes were characterized. The Ct values of these samples ranged from 17.0-29.9 for RSV, and from 20.2-34.8 for HuNoV, with some HuNoV having below the detection limit. The mean percentage of post-processing reads mapped to viral genomes was 85.1% for RSV and 40.8% for HuNoV post-capture, compared to 0.08% and 1.15% in pre-capture libraries, respectively. Full-length genomes were>99% complete in all RSV positive samples and >96% complete in 47/55 HuNoV positive samples-a significant improvement over genome recovery from pre-capture libraries. RSV transcriptome (subgenomic mRNAs) sequences were also characterized from this data. Probe-based capture enrichment offers a comprehensive approach for RSV and HuNoV genome sequencing and monitoring emerging variants.

Keywords: Respiratory syncytial virus (RSV); capture enrichment; genome sequencing; human norovirus (HuNoV).

Fig. 1.

Schematic workflow. Presented in the workflow are the different steps involved in the RSV and HuNoV capture and sequencing methodology. First row—RNA was isolated from mid-turbinate nasal swab samples (RSV) and from stool samples or infected human intestinal enteroids (HuNoV) followed by Real-Time RT-PCR to detect these viruses. Positive samples were quantified, and RNA was converted to cDNA. Second row–The cDNA was used to generate Illumina libraries with molecular barcodes and these libraries were pooled based on the Ct. values. Capture enrichment was performed with either RSV or HuNoV probe set, and enriched libraries were then sequenced on the Illumina NovaSeq 6000 instrument to generate 2×150 bp length reads. Pre-captured libraries were also sequenced followed by downstream genome reconstruction, variant, and lineage analyses.

Fig. 2.

Viral read recovery efficiency. Percent of trimmed, non-human sequence reads (post-processing) that mapped to the target viral genome in pre-capture (circles) and post-capture (triangles) libraries. CT value range of samples: ‘CT <20’ (red), ‘CT 20 to 30’ (light blue), ‘CT > 30’ (green) & ND (not detected) (pink). A: Viral reads mapping to RSV genomes, split by two subtypes. B: Viral reads mapping to HuNoV genomes, split by genotypes (GI.1, GII.4, Other GII).

Fig. 3.

Average genome coverage obtained in post-capture (triangles) and pre-capture (circles) samples. Genome reconstruction was classified as follows: ‘complete’ (within expected length range, >90% completeness & >20x coverage), ‘complete with low coverage’ (within expected length range, >90% completeness & <20x coverage), or ‘incomplete’ (below expected length range, <90% completeness & <20x coverage). CT value range of samples: ‘CT <20’ (red), ‘CT 20 to 30’ (light blue), ‘CT > 30’ (green) & ‘ND’ (pink). A: RSV samples split by RSV-A or RSV-B genotype. B: HuNoV samples split by five genotypes (GI.1, GII.4, Other GII).

Fig. 4.

The breadth of coverage for a minimal 20x coverage was calculated from the post-capture and pre-capture RSV and HuNoV libraries. Sample pairs (i.e. the same sample processed with or without capture) are shown connected by a line. Samples that could not be detected by PCR were represented with ND (not detected). The left panel represents RSV and the right panel HuNoV subgroups.

Fig. 5.

ORF expression levels in RSV-A (top panels) and RSV-B; (lower panels) pre-capture (left panels) and post-capture (right panels) samples.