Barak Noga, Fadeev Eduard, Brekhman Vera, Aharonovich Dikla, Lotan Tamar, Sher Daniel
Marine Biology Department, The Leon H. Charney School of Marine Sciences, University of Haifa, Haifa 3498838, Israel.
Department of Functional and Evolutionary Ecology, University of Vienna, 1030 Vienna, Austria.
Microorganisms. 2023 Apr 6;11(4):955. doi: 10.3390/microorganisms11040955.
Amplicon sequencing of the 16S rRNA gene is extensively used to characterize bacterial communities, including those living in association with eukaryotic hosts. Deciding which region of the 16S rRNA gene to analyze and selecting the appropriate PCR primers remains a major decision when initiating any new microbiome study. Based on a detailed literature survey of studies focusing on cnidarian microbiomes, we compared three commonly used primers targeting different hypervariable regions of the 16S rRNA gene, V1V2, V3V4, and V4V5, using the jellyfish as a model. Although all primers exhibit a similar pattern in bacterial community composition, the performance of the V3V4 primer set was superior to V1V2 and V4V5. The V1V2 primers misclassified bacteria from the Bacilli class and exhibited low classification resolution for Rickettsiales, which represent the second most abundant 16S rRNA gene sequence in all the primers. The V4V5 primer set detected almost the same community composition as the V3V4, but the ability of these primers to also amplify the eukaryotic 18S rRNA gene may hinder bacterial community observations. However, after overcoming the challenges possessed by each one of those primers, we found that all three of them show very similar bacterial community dynamics and compositions. Nevertheless, based on our results, we propose that the V3V4 primer set is potentially the most suitable for studying jellyfish-associated bacterial communities. Our results suggest that, at least for jellyfish samples, it may be feasible to directly compare microbial community estimates from different studies, each using different primers but otherwise similar experimental protocols. More generally, we recommend specifically testing different primers for each new organism or system as a prelude to large-scale 16S rRNA gene amplicon analyses, especially of previously unstudied host-microbe associations.
16S rRNA基因的扩增子测序被广泛用于表征细菌群落，包括那些与真核宿主共生的细菌群落。在启动任何新的微生物组研究时，决定分析16S rRNA基因的哪个区域以及选择合适的PCR引物仍然是一个主要决策。基于对专注于刺胞动物微生物组研究的详细文献调查，我们以水母为模型，比较了针对16S rRNA基因不同高变区的三种常用引物，即V1V2、V3V4和V4V5。尽管所有引物在细菌群落组成上表现出相似的模式，但V3V4引物组的性能优于V1V2和V4V5。V1V2引物将芽孢杆菌属的细菌错误分类，并且对立克次氏体目表现出较低的分类分辨率，而立克次氏体目在所有引物中代表第二丰富的16S rRNA基因序列。V4V5引物组检测到的群落组成与V3V4几乎相同，但这些引物扩增真核18S rRNA基因的能力可能会妨碍对细菌群落的观察。然而，在克服了每种引物所带来的挑战后，我们发现它们三者都显示出非常相似的细菌群落动态和组成。尽管如此，基于我们的结果，我们建议V3V4引物组可能最适合研究与水母相关的细菌群落。我们的结果表明，至少对于水母样本，直接比较来自不同研究的微生物群落估计值可能是可行的，每个研究使用不同的引物，但其他实验方案相似。更一般地说，我们建议在进行大规模16S rRNA基因扩增子分析之前，特别是对于以前未研究过的宿主-微生物关联，针对每个新的生物体或系统专门测试不同的引物。
