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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\u2009&lt;\u20096.4\u2009\u00d7\u200910\n                    <jats:sup>2<\/jats:sup>\n                    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\n                    <jats:italic>Toxoplasma gondii<\/jats:italic>\n                    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.\n                  <\/jats:p>","DOI":"10.1038\/s41598-021-83812-x","type":"journal-article","created":{"date-parts":[[2021,2,23]],"date-time":"2021-02-23T06:05:33Z","timestamp":1614060333000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":37,"title":["A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification"],"prefix":"10.1038","volume":"11","author":[{"given":"Xiaofang","family":"Jia","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Lvyin","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Min","family":"Wu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yun","family":"Ling","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Wei","family":"Wang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongzhou","family":"Lu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenghong","family":"Yuan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhigang","family":"Yi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaonan","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2021,2,23]]},"reference":[{"key":"83812_CR1","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1016\/S1473-3099(04)01044-8","volume":"4","author":"S Yang","year":"2004","unstructured":"Yang, S. & Rothman, R. 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