TY - JOUR
T1 - A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification
AU - Jia, Xiaofang
AU - Hu, Lvyin
AU - Wu, Min
AU - Ling, Yun
AU - Wang, Wei
AU - Lu, Hongzhou
AU - Yuan, Zhenghong
AU - Yi, Zhigang
AU - Zhang, Xiaonan
N1 - Funding Information:
The authors acknowledge funding received from the following sources: the National Science and Technology Major Project of China (2017ZX10103009-001, 2018ZX10305409-001-005), the National Natural Science Foundation of China (grant no. 81801991, 81873962, 81671998, 91542207, 91842309), and the Chinese Foundation for Hepatitis Prevention and Control, TianQing Liver Disease Research Fund Subject (TQGB20200164). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Publisher Copyright:
© 2021, The Author(s).
PY - 2021/12
Y1 - 2021/12
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85101662275&partnerID=8YFLogxK
U2 - 10.1038/s41598-021-83812-x
DO - 10.1038/s41598-021-83812-x
M3 - Short Survey/Scientific Report
C2 - 33623127
SN - 2045-2322
VL - 11
SP - 4405
JO - Scientific Reports
JF - Scientific Reports
IS - 1
M1 - 4405
ER -
