The impact of storage conditions on human stool 16S rRNA microbiome composition and diversity

Carruthers, L. V. , Moses, A., Adriko, M., Faust, C. L. , Tukahebwa, E. M., Hall, L. J., Ranford-Cartwright, L. C. and Lamberton, P. H.L. (2019) The impact of storage conditions on human stool 16S rRNA microbiome composition and diversity. PeerJ, 7, e8133. (doi:10.7717/peerj.8133)

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Abstract

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 is the accepted standard to preserve faecal 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-life physical environments found in resource-limited fieldwork conditions. Methods: Inner and outer stool samples collected from one specimen obtained from three children were stored using different storage preservation methods (raw, ethanol and RNAlater) 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, combined and aligned to the Greengenes database. Microbial diversity and composition data were generated and analysed using Quantitative Insights Into Microbial Ecology 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 in a fieldwork setting for comparative microbiome analyses should ideally be stored as consistently as possible using the same preservation method throughout.

Item Type:Articles
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Ranford-Cartwright, Dr Lisa and Carruthers, Lauren and Lamberton, Dr Poppy and Faust, Christina
Authors: Carruthers, L. V., Moses, A., Adriko, M., Faust, C. L., Tukahebwa, E. M., Hall, L. J., Ranford-Cartwright, L. C., and Lamberton, P. H.L.
College/School:College of Medical Veterinary and Life Sciences > Institute of Biodiversity Animal Health and Comparative Medicine
Journal Name:PeerJ
Publisher:PeerJ
ISSN:2167-8359
ISSN (Online):2167-8359
Copyright Holders:Copyright © 2019 Carruthers et al.
First Published:First published in PeerJ 7: e8133
Publisher Policy:Reproduced under a Creative Commons License
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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
172121Funding SchemesAnna DominiczakWellcome Trust (WELLCOTR)105614/Z/14/ZInstitute of Cardiovascular & Medical Sciences
172876SCHISTO-PERSISTPoppy LambertonEuropean Research Council (ERC)680088Institute of Biodiversity, Animal Health and Comparative Medicine