Department of Laboratory Medicine, Ren Ji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
Department of Laboratory Medicine, Shijiazhuang People's Hospital, Shijiazhuang, China.
Front Immunol. 2022 Nov 15;13:1016440. doi: 10.3389/fimmu.2022.1016440. eCollection 2022.
Metagenomic next-generation sequencing (mNGS) technology has been central in detecting infectious diseases and helping to simultaneously reveal the complex interplay between invaders and their hosts immune response characteristics. However, it needs to be rigorously assessed for clinical utility. The present study is the first to evaluate the clinical characteristics of the host DNA-removed mNGS technology for detecting SARS-CoV-2, revealing host local immune signaling and assisting genomic epidemiology.
46 swab specimens collected from COVID-19 patients were assayed by two approved commercial RT-qPCR kits and mNGS. The evolutionary tree of SARS-CoV-2 was plotted using FigTree directly from one sample. The workflow of removing the host and retaining the host was compared to investigate the influence of host DNA removal on the performances of mNGS. Functional enrichment analysis of DEGs and xCell score were used to explore the characteristics of host local immune signaling.
The detection rate of mNGS achieved 92.9% (26/28) for 28 samples with a Ct value ≤ 35 and 81.1% (30/37) for all 46 samples. The genome coverage of SARS-CoV-2 could reach up to 98.9% when the Ct value is about 20 in swab samples. Removing the host could enhance the sensitivity of mNGS for detecting SARS-CoV-2 from the swab sample but does not affect the species abundance of microbes RNA. Improving the sequencing depth did not show a positive effect on improving the detection sensitivity of SARS-CoV-2. Cell type enrichment scores found multiple immune cell types were differentially expressed between patients with high and low viral load.
The host DNA-removed mNGS has great potential utility and superior performance on comprehensive identification of SARS-CoV-2 and rapid traceability, revealing the microbiome's transcriptional profiles and host immune responses.
宏基因组下一代测序（mNGS）技术在检测传染病方面发挥了核心作用，同时有助于揭示入侵物与宿主免疫反应特征之间的复杂相互作用。然而，其临床实用性需要进行严格评估。本研究首次评估了宿主 DNA 去除 mNGS 技术检测 SARS-CoV-2 的临床特征，揭示了宿主局部免疫信号，并协助基因组流行病学研究。
采用两种经批准的商业 RT-qPCR 试剂盒和 mNGS 对 46 份来自 COVID-19 患者的拭子标本进行检测。直接从一个样本中使用 FigTree 绘制 SARS-CoV-2 的进化树。比较去除宿主和保留宿主的工作流程，以研究宿主 DNA 去除对 mNGS 性能的影响。使用差异表达基因（DEGs）的功能富集分析和 xCell 评分来探索宿主局部免疫信号的特征。
对于 Ct 值≤35 的 28 个样本，mNGS 的检测率达到 92.9%（26/28），对于所有 46 个样本，检测率达到 81.1%（30/37）。当拭子样本中的 Ct 值约为 20 时，SARS-CoV-2 的基因组覆盖度可达 98.9%。去除宿主可以提高 mNGS 从拭子样本中检测 SARS-CoV-2 的灵敏度，但不影响微生物 RNA 的物种丰度。提高测序深度对提高 SARS-CoV-2 的检测灵敏度没有积极影响。细胞类型富集评分发现，高病毒载量和低病毒载量患者之间存在多种免疫细胞类型的差异表达。
宿主 DNA 去除 mNGS 具有很大的潜在应用价值和卓越的性能，可全面识别 SARS-CoV-2，并快速追踪，揭示微生物组的转录谱和宿主免疫反应。
