Agricultural Research Service, NEA, Animal Biosciences and Biotechnology Laboratory, United States Department of Agriculture, 10300 Baltimore Avenue, B-200, Rm. 100B, BARC-East, Beltsville, MD, 20705, USA.
Agricultural Research Service, Northeast Area, Statistic Group, United States Department of Agriculture, Beltsville, MD, 20705, USA.
Sci Rep. 2021 Jun 4;11(1):11848. doi: 10.1038/s41598-021-91387-w.
We evaluated the effect of applying different sets of 16S rRNA primers on bacterial composition, diversity, and predicted function in chicken ceca. Cecal contents from Ross 708 birds at 1, 3, and 5 weeks of age were collected for DNA isolation. Eight different primer pairs targeting different variable regions of the 16S rRNA gene were employed. DNA sequences were analyzed using open-source platform QIIME2 and the Greengenes database. PICRUSt2 was used to determine the predicted function of bacterial communities. Changes in bacterial relative abundance due to 16S primers were determined by GLMs. The average PCR amplicon size ranged from 315 bp (V3) to 769 bp (V4-V6). Alpha- and beta-diversity, taxonomic composition, and predicted functions were significantly affected by the primer choice. Beta diversity analysis based on Unweighted UniFrac distance matrix showed separation of microbiota with four different clusters of bacterial communities. Based on the alpha- and beta-diversity and taxonomic composition, variable regions V1-V3(1) and (2), and V3-V4 and V3-V5 were in most consensus. Our data strongly suggest that selection of particular sets of the 16S rRNA primers can impact microbiota analysis and interpretation of results in chicken as was shown previously for humans and other animal species.
我们评估了应用不同 16S rRNA 引物对鸡盲肠中细菌组成、多样性和预测功能的影响。收集了罗斯 708 只鸡在 1、3 和 5 周龄时的盲肠内容物进行 DNA 分离。使用了 8 对针对 16S rRNA 基因不同可变区的不同引物对。使用开源平台 QIIME2 和 Greengenes 数据库分析 DNA 序列。使用 PICRUSt2 确定细菌群落的预测功能。通过 GLMs 确定 16S 引物引起的细菌相对丰度变化。PCR 扩增子的平均大小范围从 315bp（V3）到 769bp（V4-V6）。细菌多样性、分类组成和预测功能显著受引物选择的影响。基于非加权 UniFrac 距离矩阵的 beta 多样性分析显示，微生物群与四个不同的细菌群落聚类分离。基于 alpha 和 beta 多样性以及分类组成，可变区 V1-V3(1) 和 (2)、V3-V4 和 V3-V5 最为一致。我们的数据强烈表明，选择特定的 16S rRNA 引物集可以影响鸡的微生物组分析和结果解释，正如先前在人类和其他动物物种中所显示的那样。
