ena-STUDY-UNIVERSITY OF GLASGOW-07-06-2019-00:09:48:156-15
The impact of storage conditions on human stool 16S rRNA microbiome composition and diversity
Background. Multiple factors can influence stool sample integrity upon sample collection.  Preservation of faecal samples for microbiome studies is therefore an important step, particularly in tropical regions where resources are limited and high temperatures may significantly influence microbiota profiles. Freezing samples is the accepted standard to preserve samples however, cold chain methods are often unfeasible in fieldwork scenarios particularly in low and middle-income countries and alternatives are required. This study therefore aimed to address the impact of different preservative methods, time-to-freezing at ambient tropical temperatures, and stool heterogeneity on stool microbiome diversity and composition under real fieldwork conditions.   Methods. Inner, outer and mixed stool samples collected from one specimen obtained from three children, were stored using different storage preservation methods (raw, ethanol, RNAlater and OMNIgene.GUT) in a Ugandan field setting. Mixed stool was also stored using these techniques and frozen at different time-to-freezing intervals post-collection from 0 – 32 h. Metataxonomic profiling was used to profile samples, targeting the V1 – V2 regions of 16S rRNA with samples run on a Miseq platform. Reads were trimmed and combined using Python scripts and aligned to the Greengenes database. Microbial diversity and composition data were generated and analysed using Quantitative Insights Into Microbial Ecology (QIIME) and R software.  Results. Child donor was the greatest predictor of microbiome variation between the stool samples, with all samples remaining identifiable to their child of origin despite the stool being stored under a variety of conditions. However, significant differences were observed in composition and diversity between preservation techniques, but intra-preservation technique variation was minimal for all preservation methods, and across the time-to-freezing range (0 – 32 h) used. Stool heterogeneity yielded no apparent microbiome differences.  Conclusions. Stool collected for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout.
Background. Multiple factors can influence stool sample integrity upon sample collection.  Preservation of faecal samples for microbiome studies is therefore an important step, particularly in tropical regions where resources are limited and high temperatures may significantly influence microbiota profiles. Freezing samples is the accepted standard to preserve samples however, cold chain methods are often unfeasible in fieldwork scenarios particularly in low and middle-income countries and alternatives are required. This study therefore aimed to address the impact of different preservative methods, time-to-freezing at ambient tropical temperatures, and stool heterogeneity on stool microbiome diversity and composition under real fieldwork conditions.   Methods. Inner, outer and mixed stool samples collected from one specimen obtained from three children, were stored using different storage preservation methods (raw, ethanol, RNAlater and OMNIgene.GUT) in a Ugandan field setting. Mixed stool was also stored using these techniques and frozen at different time-to-freezing intervals post-collection from 0 – 32 h. Metataxonomic profiling was used to profile samples, targeting the V1 – V2 regions of 16S rRNA with samples run on a Miseq platform. Reads were trimmed and combined using Python scripts and aligned to the Greengenes database. Microbial diversity and composition data were generated and analysed using Quantitative Insights Into Microbial Ecology (QIIME) and R software.  Results. Child donor was the greatest predictor of microbiome variation between the stool samples, with all samples remaining identifiable to their child of origin despite the stool being stored under a variety of conditions. However, significant differences were observed in composition and diversity between preservation techniques, but intra-preservation technique variation was minimal for all preservation methods, and across the time-to-freezing range (0 – 32 h) used. Stool heterogeneity yielded no apparent microbiome differences.  Conclusions. Stool collected for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout.