本研究分别对新老窖泥样本的16S rDNA V4和V3-V4区进行Illumina高通量测序,比较了测序的群落多样性参数、NMDS聚类与优势门/属的相对丰度。V4区的OTUs数目(264~639)显著低于V3-V4区(459~1123);V4区的Shannon指数在新、老池壁泥中(2.47、...本研究分别对新老窖泥样本的16S rDNA V4和V3-V4区进行Illumina高通量测序,比较了测序的群落多样性参数、NMDS聚类与优势门/属的相对丰度。V4区的OTUs数目(264~639)显著低于V3-V4区(459~1123);V4区的Shannon指数在新、老池壁泥中(2.47、5.02)显著低于V3-V4区(3.33、6.46),而在新(4.98 vs 5.98)、老(5.64 vs 5.39)池底泥样本中无显著差异(p>0.05);V4区的Chao1指数在新池底泥(2455 vs 1358)、新池壁泥(1326 vs 504)和老池底泥(3475 vs 1260)中显著高于V3~V4区,在老池壁泥中无显著差异(1915 vs 1312)(p>0.05)。两个可变区的测序对NMDS聚类规律无影响。热图聚类结果显示,两个可变区的优势门/属的相对丰度很接近,V4区在属水平上更容易区分老窖池的池底泥。与V3-V4区相比,V4区更利于区分池壁泥与池底泥,且可以检测出甲烷菌的含量,更有利于窖泥菌群组成分析。本研究为16S rDNA高通量测序中V3-V4区与V4区的选择提供一定参考。展开更多
Objectives:The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using highthroughput sequencing.Methods:Saliva samples were obtained from 35 caries-free preschool children(18 bo...Objectives:The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using highthroughput sequencing.Methods:Saliva samples were obtained from 35 caries-free preschool children(18 boys and 17 girls)with primary dentition,and 16 S ribosomal DNA(r DNA)V3–V4 hypervariable regions of the microorganisms were analyzed using Illumina MiSeq.Results:At 97%similarity level,all of these reads were clustered into 334 operational taxonomic units(OTUs).Among these,five phyla(Firmicutes,Proteobacteria,Actinobacteria,Bacteroidetes,and Candidate division TM7)and13 genera(Streptococcus,Rothia,Granulicatella,Prevotella,Enterobacter,Veillonella,Neisseria,Staphylococcus,Janthinobacterium,Pseudomonas,Brevundimonas,Devosia,and Gemella)were the most dominant,constituting 99.4%and 89.9%of the salivary microbiota,respectively.The core salivary microbiome comprised nine genera(Actinomyces,Capnocytophaga,Gemella,Granulicatella,Lachnoanaerobaculum,Neisseria,Porphyromonas,Rothia,and Streptococcus).Analysis of microbial diversity and community structure revealed a similar pattern between male and female subjects.The difference in microbial community composition between them was mainly attributed to Neisseria(P=0.023).Furthermore,functional prediction revealed that the most abundant genes were related to amino acid transport and metabolism.Conclusions:Our results revealed the diversity and composition of salivary microbiota in caries-free preschool children,with little difference between male and female subjects.Identity of the core microbiome,coupled with prediction of gene function,deepens our understanding of oral microbiota in cariesfree populations and provides basic information for associating salivary microecology and oral health.展开更多
为建立用于二代测序技术的最适细菌DNA提取方法。分别用磁珠法、热裂解法、试剂盒法(有溶菌酶处理和无溶菌酶处理)、超声波法、超声+热裂解法共6种方法提取等量混合的细菌DNA,特异性扩增其16S r RNA的V3-V4高变区基因,经二代测序并分析...为建立用于二代测序技术的最适细菌DNA提取方法。分别用磁珠法、热裂解法、试剂盒法(有溶菌酶处理和无溶菌酶处理)、超声波法、超声+热裂解法共6种方法提取等量混合的细菌DNA,特异性扩增其16S r RNA的V3-V4高变区基因,经二代测序并分析结果中的细菌种类及其相对含量分布。6种方法的测序结果中,细菌种类鉴定结果无明显差异;而在磁珠法和试剂盒法(有溶菌酶处理)结果中,不同细菌的含量分布最为接近样品中的真实情况。在6种方法中,磁珠法和试剂盒法(有溶菌酶处理)适合于二代测序技术中混合细菌DNA的提取。展开更多
在组织共生微生物群系的研究中,组织样本中微生物细胞比例远低于宿主细胞,导致高通量测序数据中属于微生物的序列比例很低,不足以准确反映菌群多样性和结构组成.本文比较了HostZEROTM Microbial DNA Kit(ZYM)和NEBNext mircobiome DNA e...在组织共生微生物群系的研究中,组织样本中微生物细胞比例远低于宿主细胞,导致高通量测序数据中属于微生物的序列比例很低,不足以准确反映菌群多样性和结构组成.本文比较了HostZEROTM Microbial DNA Kit(ZYM)和NEBNext mircobiome DNA enrichment kit(NEB)两款商用DNA提取试剂盒、16S r RNA基因V3-V4高变区测序文库构建过程中的巢式PCR(NES)和扩增产物切胶回收(GEL)四种方法.与未经去除宿主DNA的方法相比,四种方法都使胃组织样本测序结果中细菌序列占比显著增加.NES和ZYM明显改变了纯菌菌株混合样本和胃组织样本菌群的组成;NEB未对菌株混合样本菌群组成造成明显影响,但对胃组织样本菌群结构影响大;只有GEL方法对样本菌群结构影响较小.综上所述,GEL方法能够去除大部分宿主DNA又对菌群组成影响最小,这为研究组织微生物群系时减少测序数据中宿主DNA的方法选择提供了参考.展开更多
文摘本研究分别对新老窖泥样本的16S rDNA V4和V3-V4区进行Illumina高通量测序,比较了测序的群落多样性参数、NMDS聚类与优势门/属的相对丰度。V4区的OTUs数目(264~639)显著低于V3-V4区(459~1123);V4区的Shannon指数在新、老池壁泥中(2.47、5.02)显著低于V3-V4区(3.33、6.46),而在新(4.98 vs 5.98)、老(5.64 vs 5.39)池底泥样本中无显著差异(p>0.05);V4区的Chao1指数在新池底泥(2455 vs 1358)、新池壁泥(1326 vs 504)和老池底泥(3475 vs 1260)中显著高于V3~V4区,在老池壁泥中无显著差异(1915 vs 1312)(p>0.05)。两个可变区的测序对NMDS聚类规律无影响。热图聚类结果显示,两个可变区的优势门/属的相对丰度很接近,V4区在属水平上更容易区分老窖池的池底泥。与V3-V4区相比,V4区更利于区分池壁泥与池底泥,且可以检测出甲烷菌的含量,更有利于窖泥菌群组成分析。本研究为16S rDNA高通量测序中V3-V4区与V4区的选择提供一定参考。
基金supported by the National Natural Science Foundation of China(No.81801028)the Zhejiang Provincial Natural Science Foundation of China(Nos.LQ19H140002 and LGF18H140004)。
文摘Objectives:The study aimed at identifying salivary microbiota in caries-free Chinese preschool children using highthroughput sequencing.Methods:Saliva samples were obtained from 35 caries-free preschool children(18 boys and 17 girls)with primary dentition,and 16 S ribosomal DNA(r DNA)V3–V4 hypervariable regions of the microorganisms were analyzed using Illumina MiSeq.Results:At 97%similarity level,all of these reads were clustered into 334 operational taxonomic units(OTUs).Among these,five phyla(Firmicutes,Proteobacteria,Actinobacteria,Bacteroidetes,and Candidate division TM7)and13 genera(Streptococcus,Rothia,Granulicatella,Prevotella,Enterobacter,Veillonella,Neisseria,Staphylococcus,Janthinobacterium,Pseudomonas,Brevundimonas,Devosia,and Gemella)were the most dominant,constituting 99.4%and 89.9%of the salivary microbiota,respectively.The core salivary microbiome comprised nine genera(Actinomyces,Capnocytophaga,Gemella,Granulicatella,Lachnoanaerobaculum,Neisseria,Porphyromonas,Rothia,and Streptococcus).Analysis of microbial diversity and community structure revealed a similar pattern between male and female subjects.The difference in microbial community composition between them was mainly attributed to Neisseria(P=0.023).Furthermore,functional prediction revealed that the most abundant genes were related to amino acid transport and metabolism.Conclusions:Our results revealed the diversity and composition of salivary microbiota in caries-free preschool children,with little difference between male and female subjects.Identity of the core microbiome,coupled with prediction of gene function,deepens our understanding of oral microbiota in cariesfree populations and provides basic information for associating salivary microecology and oral health.
文摘为建立用于二代测序技术的最适细菌DNA提取方法。分别用磁珠法、热裂解法、试剂盒法(有溶菌酶处理和无溶菌酶处理)、超声波法、超声+热裂解法共6种方法提取等量混合的细菌DNA,特异性扩增其16S r RNA的V3-V4高变区基因,经二代测序并分析结果中的细菌种类及其相对含量分布。6种方法的测序结果中,细菌种类鉴定结果无明显差异;而在磁珠法和试剂盒法(有溶菌酶处理)结果中,不同细菌的含量分布最为接近样品中的真实情况。在6种方法中,磁珠法和试剂盒法(有溶菌酶处理)适合于二代测序技术中混合细菌DNA的提取。
文摘在组织共生微生物群系的研究中,组织样本中微生物细胞比例远低于宿主细胞,导致高通量测序数据中属于微生物的序列比例很低,不足以准确反映菌群多样性和结构组成.本文比较了HostZEROTM Microbial DNA Kit(ZYM)和NEBNext mircobiome DNA enrichment kit(NEB)两款商用DNA提取试剂盒、16S r RNA基因V3-V4高变区测序文库构建过程中的巢式PCR(NES)和扩增产物切胶回收(GEL)四种方法.与未经去除宿主DNA的方法相比,四种方法都使胃组织样本测序结果中细菌序列占比显著增加.NES和ZYM明显改变了纯菌菌株混合样本和胃组织样本菌群的组成;NEB未对菌株混合样本菌群组成造成明显影响,但对胃组织样本菌群结构影响大;只有GEL方法对样本菌群结构影响较小.综上所述,GEL方法能够去除大部分宿主DNA又对菌群组成影响最小,这为研究组织微生物群系时减少测序数据中宿主DNA的方法选择提供了参考.
