Lyte Joshua M, Seyoum Mitiku M, Ayala Diana, Kers Jannigje G, Caputi Valentina, Johnson Timothy, Zhang Li, Rehberger Joshua, Zhang Guolong, Dridi Sami, Hale Brett, De Oliveira Jean E, Grum Daniel, Smith Alexandra H, Kogut Michael, Ricke Steven C, Ballou Anne, Potter Bill, Proszkowiec-Weglarz Monika
United States Department of Agriculture, Agricultural Research Service, Southeast Area, Poultry Production and Product Safety Research, Fayetteville 72701, AR, United States.
Center of Excellence for Poultry Science, University of Arkansas, Fayetteville 72701, AR, United States.
Poult Sci. 2025 Jul;104(7):105242. doi: 10.1016/j.psj.2025.105242. Epub 2025 May 1.
Bacteria are the major component of poultry gastrointestinal tract (GIT) microbiota and play an important role in host health, nutrition, physiology regulation, intestinal development, and growth. Bacterial community profiling based on the 16S ribosomal RNA (rRNA) gene amplicon sequencing approach has become the most popular method to determine the taxonomic composition and diversity of the poultry microbiota. The 16S rRNA gene profiling involves numerous steps, including sample collection and storage, DNA isolation, 16S rRNA gene primer selection, Polymerase Chain Reaction (PCR), library preparation, sequencing, raw sequencing reads processing, taxonomic classification, α- and β-diversity calculations, and statistical analysis. However, there is currently no standardized protocol for 16S rRNA gene analysis profiling and data deposition for poultry microbiota studies. Variations in DNA storage and isolation, primer design, and library preparation are known to introduce biases, affecting community structure and microbial population analysis leading to over- or under-representation of individual bacteria within communities. Additionally, different sequencing platforms, bioinformatics pipeline, and taxonomic database selection can affect classification and determination of the microbial taxa. Moreover, detailed experimental design and DNA processing and sequencing methods are often inadequately reported in poultry 16S rRNA gene sequencing studies. Consequently, poultry microbiota results are often difficult to reproduce and compare across studies. This manuscript reviews current practices in profiling poultry microbiota using 16S rRNA gene amplicon sequencing and proposes the development of guidelines for protocol for 16S rRNA gene sequencing that spans from sample collection through data deposition to achieve more reliable data comparisons across studies and allow for comparisons and/or interpretations of poultry studies conducted worldwide.
细菌是家禽胃肠道（GIT）微生物群的主要组成部分，在宿主健康、营养、生理调节、肠道发育和生长中发挥着重要作用。基于16S核糖体RNA（rRNA）基因扩增子测序方法的细菌群落分析已成为确定家禽微生物群分类组成和多样性的最常用方法。16S rRNA基因分析涉及多个步骤，包括样本采集与储存、DNA分离、16S rRNA基因引物选择、聚合酶链反应（PCR）、文库制备、测序、原始测序读段处理、分类学分类、α-和β-多样性计算以及统计分析。然而，目前在家禽微生物群研究中，尚无用于16S rRNA基因分析和数据存缴的标准化方案。已知DNA储存与分离、引物设计和文库制备方面的差异会引入偏差，影响群落结构和微生物种群分析，导致群落中个别细菌的过度或不足表征。此外，不同的测序平台、生物信息学流程和分类学数据库选择会影响微生物分类群的分类和确定。而且，在禽类16S rRNA基因测序研究中，详细的实验设计以及DNA处理和测序方法往往报道不足。因此，家禽微生物群的研究结果往往难以在不同研究中重现和比较。本文综述了目前使用16S rRNA基因扩增子测序分析家禽微生物群的方法，并建议制定从样本采集到数据存缴的16S rRNA基因测序方案指南，以实现不同研究间更可靠的数据比较，并便于对全球范围内开展的家禽研究进行比较和/或解读。
