A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification

Abstract Metagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumin...

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Autores principales:	Xiaofang Jia, Lvyin Hu, Min Wu, Yun Ling, Wei Wang, Hongzhou Lu, Zhenghong Yuan, Zhigang Yi, Xiaonan Zhang
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/a5a761cc97394abdbafba8f454340028
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spelling	oai:doaj.org-article:a5a761cc97394abdbafba8f4543400282021-12-02T16:23:14ZA streamlined clinical metagenomic sequencing protocol for rapid pathogen identification10.1038/s41598-021-83812-x2045-2322https://doaj.org/article/a5a761cc97394abdbafba8f4543400282021-02-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-83812-xhttps://doaj.org/toc/2045-2322Abstract Metagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing-based mNGS method was first developed and evaluated using a set of samples with known aetiology. Its sensitivity for RNA viruses (influenza A, H1N1) was < 6.4 × 102 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then, the rapid turnaround time of Nanopore sequencing was tested by sequencing influenza A virus and adenoviruses. Furthermore, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analysed, and the pathogens identified by Illumina sequencing showed 81.8% concordance with qPCR results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1-positive patients with meningitis/encephalitis detected HIV-1 RNA and Toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol that realizes efficient metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.Xiaofang JiaLvyin HuMin WuYun LingWei WangHongzhou LuZhenghong YuanZhigang YiXiaonan ZhangNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Xiaofang Jia Lvyin Hu Min Wu Yun Ling Wei Wang Hongzhou Lu Zhenghong Yuan Zhigang Yi Xiaonan Zhang A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification
description	Abstract Metagenomic next-generation sequencing (mNGS) holds promise as a diagnostic tool for unbiased pathogen identification and precision medicine. However, its medical utility depends largely on assay simplicity and reproducibility. In the current study, we aimed to develop a streamlined Illumina and Oxford Nanopore-based DNA/RNA library preparation protocol and rapid data analysis pipeline. The Illumina sequencing-based mNGS method was first developed and evaluated using a set of samples with known aetiology. Its sensitivity for RNA viruses (influenza A, H1N1) was < 6.4 × 102 EID50/mL, and a good correlation between viral loads and mapped reads was observed. Then, the rapid turnaround time of Nanopore sequencing was tested by sequencing influenza A virus and adenoviruses. Furthermore, 11 respiratory swabs or sputum samples pre-tested for a panel of pathogens were analysed, and the pathogens identified by Illumina sequencing showed 81.8% concordance with qPCR results. Additional sequencing of cerebrospinal fluid (CSF) samples from HIV-1-positive patients with meningitis/encephalitis detected HIV-1 RNA and Toxoplasma gondii sequences. In conclusion, we have developed a simplified protocol that realizes efficient metagenomic sequencing of a variety of clinical samples and pathogen identification in a clinically meaningful time frame.
format	article
author	Xiaofang Jia Lvyin Hu Min Wu Yun Ling Wei Wang Hongzhou Lu Zhenghong Yuan Zhigang Yi Xiaonan Zhang
author_facet	Xiaofang Jia Lvyin Hu Min Wu Yun Ling Wei Wang Hongzhou Lu Zhenghong Yuan Zhigang Yi Xiaonan Zhang
author_sort	Xiaofang Jia
title	A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification
title_short	A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification
title_full	A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification
title_fullStr	A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification
title_full_unstemmed	A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification
title_sort	streamlined clinical metagenomic sequencing protocol for rapid pathogen identification
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/a5a761cc97394abdbafba8f454340028
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A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification

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