Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island
Improving the standards for gut microbiome analysis of fecal samples: insights from the field biology of Japanese macaques on Yakushima Island
复制标题
DOI：
10.1007/s10329-018-0671-x
复制
发表时间：
2018-09-01
期刊：
影响因子：
1.7
通讯作者：
Agata, Kiyokazu
中科院分区：
文献类型：
--
作者：
Hayakawa, Takashi;Sawada, Akiko;Agata, Kiyokazu
基于粪便dna的16S核糖体RNA （rRNA）基因测序使用下一代测序仪使我们能够了解动物肠道微生物组对其特定栖息地的动态适应。粪便微生物组分析的传统技术已经在人类生物学定义的广泛背景下发展起来；因此，许多这些技术并不能立即应用于野生的非人类灵长类动物。为了建立野生动物肠道微生物组分析的标准实验方案，我们选择了屋久岛的日本猕猴（Macaca fuscata yakui）。我们测试了粪便样品处理的每个阶段的不同方案：存储、DNA提取和细菌16S rRNA基因测序区域的选择。我们还分析了圈养日本猕猴作为对照的肠道微生物组。通过对同一猕猴不同保存方法的比较，结果表明，不同保存方法（RNAlater和裂解缓冲液）产生的细菌操作分类单位（OTU）组成与标准冷冻保存方法相同，尽管每种OTU的相对丰度在数量上受到影响。检测到的细菌群的分类分配也受到测序区域的显著影响，这表明需要仔细选择测序区域和相应的16S rRNA基因的PCR引物对。本研究改进了目前野生非人灵长类动物微生物组分析的标准方法。日本猕猴被证明是理解微生物组适应各种环境的合适模型。
Fecal DNA-based 16S ribosomal RNA (rRNA) gene sequencing using next-generation sequencers allows us to understand the dynamic gut microbiome adaptation of animals to their specific habitats. Conventional techniques of fecal microbiome analysis have been developed within the broad contexts defined by human biology; hence, many of these techniques are not immediately applicable to wild nonhuman primates. In order to establish a standard experimental protocol for the analysis of the gut microbiomes of wild animals, we selected the Japanese macaques (Macaca fuscata yakui) on Yakushima Island. We tested different protocols for each stage of fecal sample processing: storage, DNA extraction, and choice of the sequencing region in the bacterial 16S rRNA gene. We also analyzed the gut microbiome of captive Japanese macaques as the control. The comparison of samples obtained from identical macaques but subjected to different protocols showed that the tested storage methods (RNAlater and lysis buffer) produced effectively the same composition of bacterial operational taxonomic units (OTUs) as the standard frozen storage method, although the relative abundance of each OTU was quantitatively affected. Taxonomic assignment of the detected bacterial groups was also significantly affected by the region being sequenced, indicating that sequencing regions and the corresponding polymerase chain reaction (PCR) primer pairs for the 16S rRNA gene should be carefully selected. This study improves the current standard methods for microbiome analysis in wild nonhuman primates. Japanese macaques were shown to be a suitable model for understanding microbiome adaptation to various environments.