Table_1_Characterizing the Human Mycobiota: A Comparison of Small Subunit rRNA, ITS1, ITS2, and Large Subunit rRNA Genomic Targets.pdf

Interest in the human microbiome has increased dramatically in the last decade. However, much of this research has focused on bacteria, while the composition and roles of their fungal counterparts remain less understood. Furthermore, a variety of methodological approaches have been applied, and the comparability between studies is unclear. This study compared four primer pairs targeting the small subunit (SSU) rRNA (18S), ITS1, ITS2, and large subunit (LSU) rRNA (26S) genomic regions for their ability to accurately characterize fungal communities typical of the human mycobiota. All four target regions of 21 individual fungal mock community taxa were capable of being amplified adequately and sequenced. Mixed mock community analyses revealed marked variability in the ability of each primer pair to accurately characterize a complex community. ITS target regions outperformed LSU and SSU. Of the ITS regions, ITS1 failed to generate sequences for Yarrowia lipolytica and all three Malassezia species when in a mixed community. These findings were further supported in studies of human sinonasal and mouse fecal samples. Based on these analyses, previous studies using ITS1, SSU, or LSU markers may omit key taxa that are identified by the ITS2 marker. Of methods commonly used in human mycobiota studies to date, we recommend selection of the ITS2 marker. Further investigation of more recently developed fungal primer options will be essential to ultimately determine the optimal methodological approach by which future human mycobiota studies ought to be standardized.

近十年来，人类微生物组（human microbiome）的研究热度显著攀升。然而，此类研究大多聚焦于细菌类群，而真菌类群的组成与功能仍有待深入解析。此外，现有研究采用了多样的实验方法，不同研究间的结果可比性尚不明确。本研究针对靶向小亚基核糖体RNA（SSU rRNA，18S）、内转录间隔区1（ITS1）、内转录间隔区2（ITS2）与大亚基核糖体RNA（LSU rRNA，26S）的4种引物对展开对比实验，评估其精准表征典型人类真菌组（mycobiota）群落的能力。针对21种单一真菌模拟群落（mock community）类群的4个目标区域，均可实现有效扩增与测序。混合模拟群落分析显示，不同引物对精准表征复杂群落的能力存在显著差异。内转录间隔区（ITS）靶区域的表现优于LSU与SSU靶区域；在ITS靶区域中，ITS1无法在混合群落中获取解脂亚罗酵母（Yarrowia lipolytica）与全部3种马拉色菌（Malassezia）的测序序列。上述结论在人类鼻窦样本与小鼠粪便样本的分析中得到进一步验证。基于本研究结果，既往采用ITS1、SSU或LSU标记的研究可能遗漏了ITS2标记可检出的关键类群。针对当前人类真菌组研究常用的方法，我们推荐选用ITS2标记。未来需针对更多新型研发的真菌引物展开研究，以确立标准化的最优实验方案，指导后续人类真菌组相关研究。