Diversity analysis and taxonomic profiles can be generated from marker-gene sequence data with the help of many available computational tools.The Quantitative Insights into Microbial Ecology Version 2(QIIME2)has been ...Diversity analysis and taxonomic profiles can be generated from marker-gene sequence data with the help of many available computational tools.The Quantitative Insights into Microbial Ecology Version 2(QIIME2)has been widely used for 16S rRNA data analysis.While many articles have demonstrated the use of QIIME2 with suitable datasets,the application to preclinical data has rarely been talked about.The issues involved in the pre-clinical data include the low-quality score and small sample size that should be addressed properly during analysis.In addition,there are few articles that discuss the detailed statistical methods behind those alpha and beta diversity significance tests that researchers are eager to find.Running the program without knowing the logic behind it is extremely risky.In this article,we first provide a guideline for analyzing 16S rRNA data using QIIME2.Then we will talk about issues in pre-clinical data,and how they could impact the outcome.Finally,we provide brief explanations of statistical methods such as group significance tests and sample size calculation.展开更多
The Ikogosi Warm Spring is a unique ecological niche in Western Nigeria with an average temperature and pH of 38°C and 5.8 respectively. It mixes with an adjacent cold spring (28°C & pH 7.6), about 100 m...The Ikogosi Warm Spring is a unique ecological niche in Western Nigeria with an average temperature and pH of 38°C and 5.8 respectively. It mixes with an adjacent cold spring (28°C & pH 7.6), about 100 meters from source, yielding a confluence body of water of 32°C and pH 7.7. To explore the bacterial community structure of this uncommon environment and to scan for potentially useful bacteria, metagenomes extracted directly from five samples (source and mid-point of warm spring;source and midpoint of cold spring, and the confluence) were analyzed. Using the MiSeq Illumina next generation sequencing protocols, the V3-V4 region of the 16S rRNA gene pool was sequenced and analyzed by QIIME (Quantitative Insights into Microbial Ecology) and R software. At least 11% (47,446) of all the sequences were unknown to any of the databases employed. Bacterial diversity and abundance at the source of both springs were extremely low, accounting for less than 0.07% of the total sequence reads at the confluence, 100 m downstream. In contrast to the highly diversified mesophilic confluence community where 21 different phyla were identified, only 4 and 5 phyla were recovered from the source-point of the warm spring and cold spring respectively. The most prevalent phyla in all samples were members of the versatile Proteobacteria (35% - 50% relative abundance), and the hardy Firmicutes (33% - 40%). Operational taxonomic units (OTUs) obtained from all the samples averaged at 1414. Temperature and pH were equally significant predictors of genomic diversity and richness, with the warm and cold spring sources having less than 5 bacteria phyla. Exiguobacterium sp. (a potential plastic degrader) and other deep rooted bacteria were found in the warm spring while the cold spring outflow contained among others such as Rubrobacter sp. and Chloroflexi sp. (which is close to the phylogenetic root of the domain Bacteria). Many taxonomically unresolved sequences could indicate the presence of potentially novel bacteria in this unique body of water and underscores the need to systematically mine these rare genetic reservoirs for biotechnological applications. Moreover, such tropical hydrothermal ecosystems could contain some unknown primitive bacteria at the origins of life.展开更多
基金S.N.Rai was partly supported with Wendell Cherry Chair in Clinical Trial Research Fund and NIH grants 5P20GM113226(CJM),1P42ES023716(PI:Sanjay Srivastava)and 1P20GM125504(PI:Richard Lamont).C.Qian was supported by the National Institutes of Health grant 5P50AA024337(CJM)and the University of Louisville Fellowship.
文摘Diversity analysis and taxonomic profiles can be generated from marker-gene sequence data with the help of many available computational tools.The Quantitative Insights into Microbial Ecology Version 2(QIIME2)has been widely used for 16S rRNA data analysis.While many articles have demonstrated the use of QIIME2 with suitable datasets,the application to preclinical data has rarely been talked about.The issues involved in the pre-clinical data include the low-quality score and small sample size that should be addressed properly during analysis.In addition,there are few articles that discuss the detailed statistical methods behind those alpha and beta diversity significance tests that researchers are eager to find.Running the program without knowing the logic behind it is extremely risky.In this article,we first provide a guideline for analyzing 16S rRNA data using QIIME2.Then we will talk about issues in pre-clinical data,and how they could impact the outcome.Finally,we provide brief explanations of statistical methods such as group significance tests and sample size calculation.
文摘The Ikogosi Warm Spring is a unique ecological niche in Western Nigeria with an average temperature and pH of 38°C and 5.8 respectively. It mixes with an adjacent cold spring (28°C & pH 7.6), about 100 meters from source, yielding a confluence body of water of 32°C and pH 7.7. To explore the bacterial community structure of this uncommon environment and to scan for potentially useful bacteria, metagenomes extracted directly from five samples (source and mid-point of warm spring;source and midpoint of cold spring, and the confluence) were analyzed. Using the MiSeq Illumina next generation sequencing protocols, the V3-V4 region of the 16S rRNA gene pool was sequenced and analyzed by QIIME (Quantitative Insights into Microbial Ecology) and R software. At least 11% (47,446) of all the sequences were unknown to any of the databases employed. Bacterial diversity and abundance at the source of both springs were extremely low, accounting for less than 0.07% of the total sequence reads at the confluence, 100 m downstream. In contrast to the highly diversified mesophilic confluence community where 21 different phyla were identified, only 4 and 5 phyla were recovered from the source-point of the warm spring and cold spring respectively. The most prevalent phyla in all samples were members of the versatile Proteobacteria (35% - 50% relative abundance), and the hardy Firmicutes (33% - 40%). Operational taxonomic units (OTUs) obtained from all the samples averaged at 1414. Temperature and pH were equally significant predictors of genomic diversity and richness, with the warm and cold spring sources having less than 5 bacteria phyla. Exiguobacterium sp. (a potential plastic degrader) and other deep rooted bacteria were found in the warm spring while the cold spring outflow contained among others such as Rubrobacter sp. and Chloroflexi sp. (which is close to the phylogenetic root of the domain Bacteria). Many taxonomically unresolved sequences could indicate the presence of potentially novel bacteria in this unique body of water and underscores the need to systematically mine these rare genetic reservoirs for biotechnological applications. Moreover, such tropical hydrothermal ecosystems could contain some unknown primitive bacteria at the origins of life.
