Clinical microbiome analysis

尽管对于微生物的认知在不断拓展，但是目前未有临床应用的案例。no microbiome-​based tests have been clinically validated for the diagnosis or treatment of disease, in part owing to an incomplete understanding of the complexity of the microbiome and its role in disease pathogenesis. 当下可期的应用在于：1）One future clinical application of microbiome analysis may be in the management and treatment of Clostridium difficile-​associated disease. 2）Another potential application of the microbiome is in the analysis of bacterial diversity, which can provide clues as to whether a patient’s illness is infectious or non-​infectious.

Human host response analyses

除了微生物的序列，很多时候能检测到人体的序列（主要介绍RNA-seq的应用）。Clinical mNGS typically focuses on microbial reads; however, there is a complementary role for the analysis of gene expression in studying human host responses to infection. 举例来说 Classification of genes by expression profiling using RNA-​seq has been used to characterize several infections, including staphylococcal bacteraemia, Lyme disease, candidiasis, tuberculosis and influenza.

Applications in oncology

可用于鉴于跟肿瘤发生有关的病原菌 In oncology, whole-​genome or directed NGS approaches to identify mutated genes can be used to simultaneously uncover viruses associated with cancer (that is, herpesviruses, papillomaviruses and polyomaviruses) and/or to gather data on virus–host interactions.

Clinical implementation of metagenomic NGS

需要有标准的操作流程和质控点，目前这个都是缺乏的。FDA给出了一些临床应用的指导意见，但是并未给出明确建议。Although the FDA has provided general guidelines for clinical validation of NGS infectious disease testing, there are no definitive recommendations for the clinical implementation of mNGS testing, nor is there mention of specific requirements.

Sensitivity and enrichment or depletion methods

在mNGS的研究中，宿主或其他的背景信号较多，为此在测序前，有必要对目标序列进行富集。第一、针对RNA而言，目前策略较为成熟，效果较理想。有1）DNase I treatment after extraction、2）the use of RNA probes followed by RNase H treatment、3）antibodies against human and mitochondrial rRNA和4）CRISPR–Cas9-based approaches等策略。第二、针对DNA而言，没有可匹配RNA效果的策略。1）根据甲基化的情况，可将目标序列富集3-5倍，Limited enrichment in the 3–5 times range can be achieved with the use of antibodies against methylated human host DNA, which enriches microbial reads owing to the lack of methylated DNA in most pathogen genomes. 2）通过细胞裂解的策略 Differential lysis of human cells followed by degradation of background DNA with DNase I—thus retaining and enriching for nucleic acid from organisms with cell walls, which include some bacteria and fungi—has been shown to provide substantial microbial enrichment of up to 1,000 times. 但裂解策略会受到众多因素影响，例如没有细胞壁，无法区分DNA来自活体还是dead microorganisms（这种区分可结合propidium monoazide策略）。第三、针对背景较丰富的情况，1）可用超高深度策略这一简单粗暴的策略，To some extent, the human host background limitation may be overcome with brute force, made possible by the increasing capacities of available sequencers. 2）利用spiked primers进行富集，可将目标序列扩增100-10000倍。Combining targeted with untargeted sequencing, the method uses variably sized panels (100–10,000) of short primers that are added (‘spiked’) into reaction mixtures to enrich for specific target organisms while retaining the breadth of metagenomic sequencing for off-​target organisms.

Laboratory workflow considerations

在样品的处理环节中，试剂、环境等不同环节中都存在微量的菌，所以1）操作过程须是有经验的人，避免污染。2）需要个数据库包含正常的环境、试剂中含有的微生物，如此在分析中排除此干扰因素。 A database of background microorganisms commonly detected in mNGS data and arising from normal flora or laboratory contamination typically needs to be maintained for accurate mNGS analyses.

Reference standards

为了mNGS在临床的应用，必须有一套标准样品，类似于ERCC。Most available metagenomic reference materials are highly customized to specific applications (for example, ZymoBIOMICS Microbial Community Standard for microbiome analyses and bacterial and fungal metagenomics) and/or focused on a more limited spectrum of organisms (for example, the National Institute of Standards and Technology (NIST) reference materials for mixed microbial DNA detection, which contain only bacteria). 这两个数据库有必要关注和研究下

Bioinformatics challenges

当下还是需要专业的人员才可以操控和分析数据，没有较为直观的可视化操作工具。另外，测序数据的量较大，数据存储、数据分析和数据安全等需综合权衡，选择是自建服务器或者购买云服务器。

分析过程软件、数据库是动态更新和发展的，在临床应用中，有必要结合特定场景固化分析过程，在充分验证基础上更新。

Cost considerations

目前的成本相对较高，未来可在试剂、测序以及生物机器人的使用上以降低成本。

Regulatory considerations

mNGS在临床上的应用，在样品到结果产出的各环节需要严格质控。