基于高通量测序技术分析人工圈养成年蜂猴的粪便微生物多样性

doi:10.12300/j.issn.1674-5817.2023.140

摘要/Abstract

摘要：

目的研究成年蜂猴的肠道菌群组成情况，以探讨人工圈养蜂猴的健康影响因素。方法采集4只成年雄性蜂猴的粪便样本，运用细菌16S rDNA通用引物扩增V4区，应用Illumina NovaSeq测序平台对蜂猴粪便微生物进行测序分析，并使用Qiime软件分析样本的复杂度和相似度。基于有效数据分析样本肠道菌群在门、属水平上的物种结构和丰度，同时应用PICRUSt软件进行菌群的代谢功能预测。结果年龄最小的蜂猴的肠道菌群多样性指数shannon高于其他蜂猴。PCoA分析显示4只蜂猴的样本菌群组成具有一定的相似度。4只蜂猴的粪便中细菌共鉴定出了12个门、18个纲、28个目、49个科、93个属和59个种。其中，优势菌门是拟杆菌门（Bacteroidetes）和厚壁菌门（Firmicutes），平均相对丰度分别为46%和28%；拟杆菌属（Bacteroides）、双歧杆菌属（Bifidobacterium）和梭菌属（Fusobacterium）的丰度较高，平均相对丰度分别为33%、6%和6%；有益菌群双歧杆菌属在4个蜂猴粪便样本中都存在，且在年龄较小的蜂猴中相对丰度最高。PICRUSt功能预测分析显示，蜂猴粪便菌群中氨基酸转运和代谢、碳水化合物转运和代谢等功能基因的丰度相对较高。结论应用Illumina NovaSeq高通量测序技术可以较全面地检测蜂猴的粪便菌群。蜂猴粪便中细菌组成具有丰富的多样性，其中还有许多未被分类鉴定且相对丰度较高的细菌，需要进一步研究。

关键词: 蜂猴, 粪便, 高通量测序, 优势菌群, 功能预测

Abstract:

Objective To analyze the composition of microbial community in gut of elderly male Nycticebus bengalensis, aiming to explore the health impact factors associated with artificial captivity. Methods Fecal samples of 4 adult male Nycticebus bengalensis were collected and the V4 region was amplified using bacterial 16S rDNA universal primers. Illumina NovaSeq sequencing platform was used for microbial sequencing analysis. The complexity and similarity of the samples were analyzed using the QIIME software analysis tool. The species structure and abundance of the intestinal flora were analyzed at the phylum and genus levels based on validated data. The PICRUSt software was applied to predict the metabolic function of the flora. Results The results showed that the diversity index (Shannon index) of the flora in the youngest Nycticebus bengalensis was higher than that of the others. PCoA analysis showed that the bacterial community compositions of the four samples had a certain degree of similarity. Bacteria identified in the Nycticebus bengalensis feces included 12 phyla, 18 classes, 28 orders, 49 families, 93 genera, and 59 species. Among them, the dominant phyla were Bacteroidetes and Firmicutes with average relative abundances of 46% and 28%, respectively; The genera Bacteroides, Bifidobacterium, and Fusobacterium had higher abundances, with relative abundances of 33%, 6%, and 6%, respectively; The beneficial genus Bifidobacterium was found in all samples, with the highest relative abundance found in the younger Nycticebus bengalensis. PICRUSt function prediction analysis showed that the abundance of the functional genes related to amino acid transport and metabolism, carbohydrate transport and metabolism,was relatively high. Conclusion The use of Illumina NovaSeq high-throughput sequencing technology can comprehensively detect the fecal microbial community of the Nycticebus bengalensis. The bacterial composition of Nycticebus bengalensis has rich diversity. Many bacteria are not identified and have higher relative abundance, which need further study.

Key words: Nycticebus bengalensis, Feces, High-throughput sequencing, Dominant bacteria, Function prediction

中图分类号:

R-332

张飞燕,刘超,吕龙宝. 基于高通量测序技术分析人工圈养成年蜂猴的粪便微生物多样性[J]. 实验动物与比较医学, 2024, 44(3): 297-304. DOI: 10.12300/j.issn.1674-5817.2023.140.

Feiyan ZHANG,Chao LIU,Longbao Lü. Analysis of Microbial Diversity in Feces of Adult Nycticebus bengalensis by High-throughput Sequencing[J]. Laboratory Animal and Comparative Medicine, 2024, 44(3): 297-304. DOI: 10.12300/j.issn.1674-5817.2023.140.

图/表 4

图1 蜂猴粪便样本中菌群的复杂性分析注：A为基于OTUs的维恩图（每个圈代表一个样本，圈和圈重叠部分的数字代表样本之间共有的OTUs个数，没有重叠部分的数字代表样本的特有OTUs个数）；B为观察指数稀释曲线；C为Beta多样性指数热图。N1、N2、N3、N4为4只蜂猴的样本编号，N1，N2和N4为12岁，N3为8岁。

Figure 1 Analysis of the complexity of microbial communities in fecal samples of Nycticebus bengalensisNote： A， Venn diagram based on OTUs （Each circle represents a sample， the numbers in overlapping circles represents the number of OTUs shared between samples， and the numbers in non-overlapping parts represent the number of unique OTUs of the samples）； B， Rarefaction curves of observed index； C， Beta diversity index heatmap. N1, N2, N3, and N4 are the sample numbers for the 4 Nycticebus bengalensis. N1， N2， and N4 were 12-year-old Nycticebus bengalensis and N3 was an 8-year-old Nycticebus bengalensis.

表1 4只蜂猴的粪便样本中微生物Alpha多样性指数统计表

Table 1 Statistical table of microbial Alpha diversity indices in the fecal samples of 4 Nycticebus bengalensis

Alpha多样性指数 Alpha diversity indices	蜂猴粪便样本编号 Fecal sample number of Nycticebus bengalensis				平均值±标准误 Mean±standard error
Alpha多样性指数 Alpha diversity indices	N1	N2	N3	N4	平均值±标准误 Mean±standard error
Observed species	260.00	243.00	256.00	243.00	250.50±8.81
shannon	5.30	5.39	5.75	4.95	5.35±0.33
simpson	0.95	0.96	0.96	0.93	0.95±0.02
chao1	275.83	248.67	259.44	269.64	263.39±11.92
ACE	270.28	251.79	260.40	274.87	264.33±10.31
goods_coverage	1.00	1.00	1.00	1.00	1.00±0.00

图2 蜂猴粪便样本中菌群在门（A）、属（B）水平上的物种相对丰度注：N1、N2、N3、N4为4只蜂猴的样本编号，N1，N2和N4为12岁蜂猴，N3为8岁蜂猴。

Figure 2 Relative abundance of microbial communities in Nycticebus bengalensis fecal samples at the phylum （A） and genus （B） levelsNote：N1， N2， N3， and N4 were the sample numbers of the 4 Nycticebus bengalensis. N1， N2， and N4 were 12-year-old Nycticebus bengalensis and N3 was an 8-year-old Nycticebus bengalensis.

图3 蜂猴粪便样本中肠道微生物菌群的功能分析注：A，PICRUSt功能注释聚类热图；B，KEGG数据库预测结果统计图。

Figure 3 Functional analysis of the gut microbial communities in Nycticebus bengalensis fecal samplesNote： A， PICRUSt functional annotation clustering heatmap； B，Statistical chart of predicted results from KEGG database.

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