RAPIDprep: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples
Author:
Tulloch Rachel L.12ORCID, Kim Karan12, Sikazwe Chisha34ORCID, Michie Alice34, Burrell Rebecca25ORCID, Holmes Edward C.2ORCID, Dwyer Dominic E.26ORCID, Britton Philip N.25, Kok Jen6ORCID, Eden John-Sebastian12ORCID
Affiliation:
1. Centre for Virus Research, Westmead Institute for Medical Research, Westmead, NSW 2145, Australia 2. Sydney Institute for Infectious Diseases, School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, Australia 3. PathWest Laboratory Medicine WA, Department of Microbiology, Nedlands, WA 6009, Australia 4. School of Biomedical Sciences, The University of Western Australia, Crawley, WA 6009, Australia 5. Departments of Infectious Diseases and Microbiology, The Children’s Hospital at Westmead, Westmead, NSW 2145, Australia 6. NSW Health Pathology Institute for Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW 2145, Australia
Abstract
Emerging infectious disease threats require rapid response tools to inform diagnostics, treatment, and outbreak control. RNA-based metagenomics offers this; however, most approaches are time-consuming and laborious. Here, we present a simple and fast protocol, the RAPIDprep assay, with the aim of providing a cause-agnostic laboratory diagnosis of infection within 24 h of sample collection by sequencing ribosomal RNA-depleted total RNA. The method is based on the synthesis and amplification of double-stranded cDNA followed by short-read sequencing, with minimal handling and clean-up steps to improve processing time. The approach was optimized and applied to a range of clinical respiratory samples to demonstrate diagnostic and quantitative performance. Our results showed robust depletion of both human and microbial rRNA, and library amplification across different sample types, qualities, and extraction kits using a single workflow without input nucleic-acid quantification or quality assessment. Furthermore, we demonstrated the genomic yield of both known and undiagnosed pathogens with complete genomes recovered in most cases to inform molecular epidemiological investigations and vaccine design. The RAPIDprep assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations.
Funder
Snow Medical Foundation BEAT COVID-19 research program National Health and Medical Research Council Centre of Research Excellence in Emerging Infectious Diseases Medical Research Future Fund
Subject
Virology,Infectious Diseases
Reference46 articles.
1. Forty Years of Molecular Diagnostics for Infectious Diseases;Schmitz;J. Clin. Microbiol.,2022 2. Novel Hendra Virus Variant Detected by Sentinel Surveillance of Horses in Australia;Annand;Emerg. Infect. Dis.,2022 3. A new coronavirus associated with human respiratory disease in China;Wu;Nature,2020 4. Zhang, D., Lou, X., Yan, H., Pan, J., Mao, H., Tang, H., Shu, Y., Zhao, Y., Liu, L., and Li, J. (2018). Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples. BMC Genom., 19. 5. Shi, M., Zhao, S., Yu, B., Wu, W.-C., Hu, Y., Tian, J.-H., Yin, W., Ni, F., Hu, H.-L., and Geng, S. (2022). Total infectome characterization of respiratory infections in pre-COVID-19 Wuhan, China. PLoS Pathog., 18.
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