preprints.org > biology and life sciences > virology > doi: 10.20944/preprints202212.0417.v1

Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Version 1 : Received: 21 December 2022 / Approved: 22 December 2022 / Online: 22 December 2022 (04:25:19 CET)

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 to provide cause agnostic laboratory diagnosis of infection within 24 hours 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 protocol without input nucleic acid quantification or quality assessment. Furthermore, we demonstrate 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 towards integration of modern genomic techniques to infectious disease investigations.

RNA sequencing; metagenomics; infectious diseases; diagnostics

Biology and Life Sciences, Virology

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