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

Doddapaneni, H.; Bhamidipati, S. V.; Surathu, A.; Chao, H.; Paiva Agustinho, D.; Xiang, Q.; Kottapalli, K.; Santhanam, A.; Momin, Z.; Walker, K.; Menon, V. K.; Weissenberger, G. M.; Emerick, N.; Mahjabeen, F.; Meng, Q.; Hu, J.; Sucgang, R.; Henke, D. M.; Sedlazeck, F.; Khan, Z.; Metcalf, G. A.; Avadhanula, V.; Piedra, P. A.; Ramani, S.; Atmar, R. L.; Estes, M. K.; Petrosino, J. F.; Gibbs, R.; Muzny, D.; Javornik Cregeen, S. J.

doi:10.1101/2024.09.16.613242

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bioRxiv · 10.1101/2024.09.16.613242

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

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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. IMPORTANCERespiratory syncytial virus (RSV) and human noroviruses (HuNoV) are NIAID category C and category B priority pathogens, respectively, that inflict significant health consequences on children, adults, immunocompromised patients, and the elderly. Due to the high strain diversity of RSV and HuNoV genomes, obtaining complete genomes to monitor viral evolution and pathogenesis is challenging. In this paper, we present the design, optimization, and benchmarking of a comprehensive oligonucleotide target capture method for these pathogens. All 85 RSV samples and 49/55 HuNoV samples were patient-derived with six human intestinal enteroids. The methodology described here results has a higher success rate in obtaining full-length RSV and HuNoV genomes, enhancing the efficiency of studying these viruses and mutations directly from patient-derived samples.

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Doddapaneni, H., Bhamidipati, S. V., Surathu, A., Chao, H., Paiva Agustinho, D., Xiang, Q., Kottapalli, K., Santhanam, A., Momin, Z., Walker, K., Menon, V. K., Weissenberger, G. M., Emerick, N., Mahjabeen, F., Meng, Q., Hu, J., Sucgang, R., Henke, D. M., Sedlazeck, F., Khan, Z., Metcalf, G. A., Avadhanula, V., Piedra, P. A., Ramani, S., Atmar, R. L., Estes, M. K., Petrosino, J. F., Gibbs, R., Muzny, D., Javornik Cregeen, S. J.. 2024-09-16. Complete Genomic Characterization of Global Pathogens, Respiratory Syncytial Virus (RSV), and Human Norovirus (HuNoV) Using Probe-based Capture Enrichment.. https://doi.org/10.1101/2024.09.16.613242

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