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A rapid bacterial pathogen and antimicrobial resistance diagnosis workflow using Oxford nanopore adaptive sequencing method

纳米孔测序 基因组 计算生物学 DNA测序 纳米孔 生物 工作流程 病菌 基因 DNA 遗传学 计算机科学 纳米技术 材料科学 数据库
作者
Hang Cheng,Yanfa Sun,Qing Yang,Minggui Deng,Zhijian Yu,Gang Zhu,Jiuxin Qu,Lei Liu,Liang Yang,Yu Xia
出处
期刊:Briefings in Bioinformatics [Oxford University Press]
卷期号:23 (6) 被引量:14
标识
DOI:10.1093/bib/bbac453
摘要

Abstract Metagenomic sequencing analysis (mNGS) has been implemented as an alternative approach for pathogen diagnosis in recent years, which is independent of cultivation and is able to identify all potential antibiotic resistance genes (ARGs). However, current mNGS methods have to deal with low amounts of prokaryotic deoxyribonucleic acid (DNA) and high amounts of host DNA in clinical samples, which significantly decrease the overall microbial detection resolution. The recently released nanopore adaptive sampling (NAS) technology facilitates immediate mapping of individual nucleotides to a given reference as each molecule is sequenced. User-defined thresholds allow for the retention or rejection of specific molecules, informed by the real-time reference mapping results, as they are physically passing through a given sequencing nanopore. We developed a metagenomics workflow for ultra-sensitive diagnosis of bacterial pathogens and ARGs from clinical samples, which is based on the efficient selective ‘human host depletion’ NAS sequencing, real-time species identification and species-specific resistance gene prediction. Our method increased the microbial sequence yield at least 8-fold in all 21 sequenced clinical Bronchoalveolar Lavage Fluid (BALF) samples (4.5 h from sample to result) and accurately detected the ARGs at species level. The species-level positive percent agreement between metagenomic sequencing and laboratory culturing was 100% (16/16) and negative percent agreement was 100% (5/5) in our approach. Further work is required for a more robust validation of our approach with large sample size to allow its application to other infection types.
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