高通量测序技术分析无特定病原体级实验小鼠肠道的菌群组成

Analysis of intestinal microbial composition in specific pathogen free mice using high-throughput sequencing

目的:应用高通量测序技术检测无特定病原体(specific pathogen free, SPF)级实验小鼠肠道的菌群组成,分析其细菌携带状况。方法:应用高通量测序技术,对14个品系共65只SPF级实验小鼠的回盲部肠道内容物样本中细菌16S r RNA V3~V4区进行深度测序,分析样本的菌群组成、丰度和Alpha多样性,并在属水平上筛选葡萄球菌属、假单胞菌属、支原体和棒状杆菌属。结果:65只SPF级健康成年小鼠的肠道内容物样本经数据过滤和质控后,共得到144 318条高质量序列用于后续分析,结果提示相同品系样品间菌群丰度表现出明显的差异。所有样本的序列主要集中在以下8个门,依次为厚壁菌门(Firmicutes)、拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、疣微菌门(Verrucomicrobia)、放线菌门(Actinobacteria)、Saccharibacteria、软壁菌门(Tenericutes)和螺旋菌门(Spirochaetae)。在属水平上检测到了葡萄球菌属、假单胞菌属、支原体和棒状杆菌属。结论:相同品系的SPF级实验小鼠肠道菌群在物种组成、丰度及Alpha多样性方面也会表现出显著差异,有必要致力于研究各种方法以使实验动物的肠道菌群在可控和可监测范围内,高通量测序技术提供了这一可能。

Objective: To analyze the intestinal microbial composition in specific pathogen free(SPF) mice using highthroughput sequencing. Methods: A total of 65 SPF mice of 14 strains were collected. Samples of fresh ileocecus content from the SPF mice were examined using high-throughput sequencing, and 16 S rRNA V3-V4 variable regions of bacterium were deeply sequenced. The composition, alpha diversity, and the abundance of microbial flora in ileocecus content were explored, while Staphylococcus, Pseudomonas, Mycoplasma and Corynebacterium were identified at the genus level. Results: Altogether 144 318 high-quality genome sequences were obtained after data filter and quality control. Remarkable differences were found in Alpha diversity(species richness, diversity and evenness) of the microbial composition between samples from mice of the same strains. At the phylum level, the main sequences were largely concentrated as follows:Firmicutes, Bacteroidetes, Proteobacteria, Verrucomicrobia, Actinobacteria, Saccharibacteria, Tenericutes and Spirochaetae.At the genus level, Staphylococcus, Pseudomonas, Anaeroplasma and Corynebacterium were detected in some samples.Conclusions: The composition, alpha diversity and the abundance of the microbial flora in ileocecus contents are substantially different in mice of same strain. There is a need to keep intestinal microbial composition in SPF mice under control,and high-throughput sequencing may offer such possibility.