A rapid bacterial pathogen and antimicrobial resistance diagnosis workflow using Oxford nanopore adaptive sequencing method
A rapid bacterial pathogen and antimicrobial resistance diagnosis workflow using Oxford nanopore adaptive sequencing method
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DOI：
10.1093/bib/bbac453
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发表时间：
2022-10-18
影响因子：
9.5
通讯作者：
Xia, Yu
中科院分区：
文献类型：
--
作者：
Cheng, Hang;Sun, Yuhong;Xia, Yu
宏基因组测序分析（mNGS）是近年来发展起来的一种新的病原体诊断方法，它不依赖于培养，能够识别所有潜在的抗生素耐药基因（ARG）。然而，目前的mNGS方法必须处理临床样品中低含量的原核脱氧核糖核酸（DNA）和高含量的宿主DNA，这显著降低了整体微生物检测分辨率。最近发布的纳米孔自适应采样（NAS）技术有助于在每个分子测序时立即将单个核苷酸映射到给定的参考。用户定义的阈值允许保留或拒绝特定分子，由实时参考映射结果通知，因为它们物理地通过给定的测序纳米孔。我们开发了一种宏基因组学工作流程，用于临床样本中细菌病原体和ARG的超灵敏诊断，该工作流程基于高效的选择性“人类宿主耗竭”NAS测序、实时物种鉴定和物种特异性耐药基因预测。我们的方法在所有21个测序的临床支气管肺泡灌洗液（BALF）样本中（从样本到结果的4.5小时）将微生物序列产率提高了至少8倍，并在物种水平上准确地检测了ARG。在我们的方法中，宏基因组测序和实验室培养之间的物种水平的阳性百分比一致性为100%（16/16），阴性百分比一致性为100%（5/5）。需要进一步的工作来更强大的验证我们的方法与大样本量，使其应用于其他感染类型。
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.