RAPID<i>prep</i>: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples
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 RAPID<i>prep</i> assay,...
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| Format: | Article |
| Language: | English |
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MDPI AG
2023-04-01
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| Series: | Viruses |
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| Online Access: | https://www.mdpi.com/1999-4915/15/4/1006 |
| _version_ | 1797603147165728768 |
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| author | Rachel L. Tulloch Karan Kim Chisha Sikazwe Alice Michie Rebecca Burrell Edward C. Holmes Dominic E. Dwyer Philip N. Britton Jen Kok John-Sebastian Eden |
| author_facet | Rachel L. Tulloch Karan Kim Chisha Sikazwe Alice Michie Rebecca Burrell Edward C. Holmes Dominic E. Dwyer Philip N. Britton Jen Kok John-Sebastian Eden |
| author_sort | Rachel L. Tulloch |
| collection | DOAJ |
| description | 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 RAPID<i>prep</i> 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 RAPID<i>prep</i> assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations. |
| first_indexed | 2024-03-11T04:26:20Z |
| format | Article |
| id | doaj.art-985eda2126524971807131c30a755b33 |
| institution | Directory Open Access Journal |
| issn | 1999-4915 |
| language | English |
| last_indexed | 2024-03-11T04:26:20Z |
| publishDate | 2023-04-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Viruses |
| spelling | doaj.art-985eda2126524971807131c30a755b332023-11-17T21:46:52ZengMDPI AGViruses1999-49152023-04-01154100610.3390/v15041006RAPID<i>prep</i>: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical SamplesRachel L. Tulloch0Karan Kim1Chisha Sikazwe2Alice Michie3Rebecca Burrell4Edward C. Holmes5Dominic E. Dwyer6Philip N. Britton7Jen Kok8John-Sebastian Eden9Centre for Virus Research, Westmead Institute for Medical Research, Westmead, NSW 2145, AustraliaCentre for Virus Research, Westmead Institute for Medical Research, Westmead, NSW 2145, AustraliaPathWest Laboratory Medicine WA, Department of Microbiology, Nedlands, WA 6009, AustraliaPathWest Laboratory Medicine WA, Department of Microbiology, Nedlands, WA 6009, AustraliaSydney Institute for Infectious Diseases, School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, AustraliaSydney Institute for Infectious Diseases, School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, AustraliaSydney Institute for Infectious Diseases, School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, AustraliaSydney Institute for Infectious Diseases, School of Medical Sciences, The University of Sydney, Sydney, NSW 2006, AustraliaNSW Health Pathology Institute for Clinical Pathology and Medical Research, Westmead Hospital, Westmead, NSW 2145, AustraliaCentre for Virus Research, Westmead Institute for Medical Research, Westmead, NSW 2145, AustraliaEmerging 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 RAPID<i>prep</i> 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 RAPID<i>prep</i> assay is a simple and effective tool, and representative of an important shift toward the integration of modern genomic techniques with infectious disease investigations.https://www.mdpi.com/1999-4915/15/4/1006RNA sequencingmetagenomicsinfectious diseasesdiagnostics |
| spellingShingle | Rachel L. Tulloch Karan Kim Chisha Sikazwe Alice Michie Rebecca Burrell Edward C. Holmes Dominic E. Dwyer Philip N. Britton Jen Kok John-Sebastian Eden RAPID<i>prep</i>: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples Viruses RNA sequencing metagenomics infectious diseases diagnostics |
| title | RAPID<i>prep</i>: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples |
| title_full | RAPID<i>prep</i>: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples |
| title_fullStr | RAPID<i>prep</i>: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples |
| title_full_unstemmed | RAPID<i>prep</i>: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples |
| title_short | RAPID<i>prep</i>: A Simple, Fast Protocol for RNA Metagenomic Sequencing of Clinical Samples |
| title_sort | rapid i prep i a simple fast protocol for rna metagenomic sequencing of clinical samples |
| topic | RNA sequencing metagenomics infectious diseases diagnostics |
| url | https://www.mdpi.com/1999-4915/15/4/1006 |
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