This review provides a state-of-the-art description of the performance of Sanger cycle sequencing of the 16S rRNA gene for routine identification of bacteria in the clinical microbiology laboratory. A detailed description of the technology and current methodology is outlined with a major focus on proper data analyses and interpretation of sequences. The remainder of the article is focused on a comprehensive evaluation of the application of this method for identification of bacterial pathogens based on analyses of 16S multialignment sequences. In particular, the existing limitations of similarity within 16S for genus- and species-level differentiation of clinically relevant pathogens and the lack of sequence data currently available in public databases is highlighted. A multiyear experience is described of a large regional clinical microbiology service with direct 16S broad-range PCR followed by cycle sequencing for direct detection of pathogens in appropriate clinical samples. The ability of proteomics (matrix-assisted desorption ionization-time of flight) versus 16S sequencing for bacterial identification and genotyping is compared. Finally, the potential for whole-genome analysis by next-generation sequencing (NGS) to replace 16S sequencing for routine diagnostic use is presented for several applications, including the barriers that must be overcome to fully implement newer genomic methods in clinical microbiology. A future challenge for large clinical, reference, and research laboratories, as well as for industry, will be the translation of vast amounts of accrued NGS microbial data into convenient algorithm testing schemes for various applications (i.e., microbial identification, genotyping, and metagenomics and microbiome analyses) so that clinically relevant information can be reported to physicians in a format that is understood and actionable. These challenges will not be faced by clinical microbiologists alone but by every scientist involved in a domain where natural diversity of genes and gene sequences plays a critical role in disease, health, pathogenicity, epidemiology, and other aspects of life-forms. Overcoming these challenges will require global multidisciplinary efforts across fields that do not normally interact with the clinical arena to make vast amounts of sequencing data clinically interpretable and actionable at the bedside.

中文翻译：

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16S rRNA 基因循环测序在临床微生物实验室常规细菌鉴定中的性能和应用。

本综述提供了对 16S rRNA 基因桑格循环测序性能的最新技术描述，用于临床微生物实验室中细菌的常规鉴定。概述了该技术和当前方法的详细描述，主要关注正确的数据分析和序列的解释。本文的其余部分侧重于对基于 16S 多重比对序列分析的该方法在细菌病原体鉴定中的应用的综合评价。特别是，16S 内相似性在临床相关病原体的属和种水平分化方面的现有局限性以及公共数据库中目前可用的序列数据的缺乏都受到了强调。描述了大型区域临床微生物学服务的多年经验，该服务采用直接 16S 广域 PCR，然后进行循环测序，直接检测适当临床样本中的病原体。蛋白质组学（基质辅助解吸电离飞行时间）与 16S 测序在细菌鉴定和基因分型方面的能力进行了比较。最后，通过下一代测序 (NGS) 进行全基因组分析取代 16S 测序用于常规诊断用途的潜力可用于多种应用，包括在临床微生物学中全面实施更新的基因组方法必须克服的障碍。大型临床、参考和研究实验室以及工业的未来挑战，将大量累积的 NGS 微生物数据转换为适用于各种应用（即微生物鉴定、基因分型、宏基因组学和微生物组分析）的便捷算法测试方案，以便可以以可理解的格式向医生报告临床相关信息和可操作的。这些挑战不仅是临床微生物学家面临的，而且是涉及基因和基因序列的自然多样性在疾病、健康、致病性、流行病学和生命形式的其他方面发挥关键作用的领域的每一位科学家。克服这些挑战将需要跨领域的全球多学科努力，这些领域通常不会与临床领域相互作用，以使大量测序数据在临床上可解释并在床边可操作。