A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification.
A streamlined clinical metagenomic sequencing protocol for rapid pathogen identification.
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用于快速病原体识别的简化临床宏基因组测序方案
DOI：
10.1038/s41598-021-83812-x
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发表时间：
2021-02-23
影响因子：
4.6
通讯作者：
Zhang X
中科院分区：
文献类型：
--
作者：
Jia X;Hu L;Wu M;Ling Y;Wang W;Lu H;Yuan Z;Yi Z;Zhang X
元基因组下一代测序(MNGS)有望成为无偏见病原体鉴定和精确医学的诊断工具。然而，它的医学用途在很大程度上取决于检测的简单性和重复性。在目前的研究中，我们的目标是开发一种简化的基于Illumina和牛津纳米孔的DNA/RNA文库制备方案和快速数据分析流水线。首次开发了基于Illumina测序的mNGS方法，并使用一组已知病因学的样本进行了评估。其对甲型H1N1流感病毒的敏感度为 <； 6.4 × 102EID50/mL，病毒载量与MAP读数之间有良好的相关性。然后，通过对甲型流感病毒和腺病毒进行测序，测试了纳米孔测序的快速周转时间。此外，对11份呼吸道拭子或痰标本进行了一组病原体的预检测，Illumina测序结果与qPCR结果的符合率为81.8%。对HIV-1阳性脑膜炎/脑炎患者的脑脊液(CSF)样本进行额外测序，检测到HIV-1RNA和弓形虫序列。总之，我们开发了一种简化的方案，实现了在具有临床意义的时间框架内对各种临床样本和病原体进行有效的元基因组测序。
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 × 102EID50/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 andToxoplasma gondiisequences. 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.
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