病原宏基因组高通量测序性能确认方案

The scheme for validation of clinical metagenomics sequencing assay

宏基因组学利用新一代高通量测序技术,以特定环境下病原体基因组为研究对象,在分析病原体多样性、种群结构、进化关系的基础上,进一步探究病原体的群体功能活性、相互作用及其与环境之间的关系,发掘潜在的生物学意义。目前,绝大部分的宏基因组学研究都集中在临床价值评价,宏基因组检测临床应用前分析性能确认的研究相对空白,北京协和医院检验科研究团队结合多年病原宏基因组检测的经验和国内外相关研究成果,就病原宏基因组项目医院本地化开展前的性能确认工作,从临床预期用途、方法学建立、性能确认、标准操作作业书4个方面提出了具体的实施方案,具体包含:标本类型和病原体范围、生物信息流程建立、生物信息分析参考盘制备和评估、湿实验流程的建立、背景核酸数据模型的建立、参考盘制备和全流程的性能确认等。

Clinical metagenomic next-generation sequencing(mNGS)entails unbiased shotgun sequencing of all microbial and host nucleic acids present in a clinical sample.By analyzing the microbiota diversity,taxonomic,and phylogenetic relationships of clinical specimens,metagenomics related analysis provides an opportunity to investigate substantial biological significance of different microbes.According to the published paper,most studies on mNGS mainly focused on the clinical impact evaluation.However,the studies focused on the analytical performance validation of mNGS before clinical application were rare.Here,a scheme,included intended use,method establishment,assay validation and standard operating protocol,for the laboratory validation of clinical metagenomics sequencing assay was provided by summarizing experiences of clinical laboratory department of Peking Union Medical College Hospital protocol and relevant research.In this scheme,we discussed important topics of mNGS laboratory validation as below:specimen type and pathogen list,bioinformatics pipeline setup,dry lab standard preparation and performance validation,mNGS workflow setup,background nucleotide acid evaluation,wet lab standard preparation and performance validation.