基于Illumina MiSeq的麋鹿粪样菌群多样性分析

Bacteria Community Diversity of Feces of Père David’s Deer Analyzed by Illumina MiSeq

为探究麋鹿(Elaphurus davidianus)肠道菌群的结构和组成,为麋鹿消化道疾病防治提供有价值的基础数据,应用Illumina MiSeq对4个麋鹿栖息地32只麋鹿粪便样本的16S rRNA的V3—V4可变区进行扩增,结合样本的OTU种类及丰度,用R软件进行聚类和主成分分析(PCA),并计算多样性指数。结果表明:共获得1 438 677条有效序列,平均每个样品有(44 959±12 153)条有效序列;将序列拼接优化,在97%相似度条件下获得31 459个物种分类的OTUs,平均每个样品为(983±240)个OTUs;平均读长为426 bp。对测序数据注释后共获得11个门和74个属,在门水平上,厚壁菌门(Firmicutes)和拟杆菌门(Bacteroidetes)为优势菌群;优势菌属(相对丰度大于1%)一共有15种,占89%。Alpha多样性物种指数范围区间较大,香农指数为2.42~5.83,辛普森指数为0.007 3~0.148 6,表明不同麋鹿的细菌多样性存在差异。运用主成分分析,将4地的麋鹿粪便样品聚为3个群落,其中石首麋鹿国家级自然保护区、大部分辽宁辽阳千山鹿场样品分别聚在一起,而北京麋鹿生态实验中心、河北滦河上游国家级自然保护区和少量辽宁辽阳千山鹿场样品聚在一起。研究表明,随麋鹿迁出时间的延长,麋鹿肠道菌群聚类分析的远近越明显,可能与迁出地的麋鹿饮食改变有关。

In order to explore the diversity and composition of microbiota in the feces of Père David’s deer(Elaphurus davidianus) and provide valuable basic data for the study of Père David’s deer digestive tract diseases, Illumina MiSeq was used to amplify the V3-V4 variable region of 16S rRNA from 32 Père David’s deer feces samples in four Père David’s deer habitats. In combination with the OTU types and abundance of the samples, R software was used to generate the corresponding cluster heat map and principal component analysis(PCA) map, and the diversity index was calculated at the same time. The results showed that 1,438,677 sequences were detected by high-throughput sequencing, with an average of(44,959±12,153) effective sequences per sample. After sequence splicing optimization, 31,459 species classification OTUs were obtained under the condition of 97% similarity, with an average of(983±240) OTUs per sample. The average read length is 426 bp. At the phylum grading level, the bacteria came mainly from 11 phyla, 74 genera. Firmicutes and Bacteroidetes were the dominant bacteria. There are 15 dominant genera(relative abundance greater than 1%), accounting for 89%. The range of Alpha diversity species index is large, ranged from 2. 42 to 5. 83 for Shannon index and 0. 0073to 0. 1486 for Simpson index, indicating that there were differences in bacterial diversity among different Père David’s deer.In the principal component analysis, all fecal samples were divided into three parts, in which samples from Shishou Milu National Nature Reserve and from most of Liaoning Liaoyang Qianshan Deer Farm were clustered together, while samples from Beijing Milu Ecological Research Center, from the upstream of Luanhe River Nature Reserve in Hebei Province, and from Liaoning Liaoyang Qianshan Deer Farm were clustered together. The results suggested that the cluster analysis of intestinal microflora of Père David’s deer was more separated with the increased migration time of Père David’s deer, which might be related to changes in food composition of Père David’s deer at different areas.