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

Lauren V. Carruthers*, Arinaitwe Moses, Moses Adriko, Christina L. Faust, Edridah M. Tukahebwa, Lindsay J. Hall, Lisa C. Ranford-Cartwright, Poppy H.L. Lamberton

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

18 Citations (Scopus)

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.

Original languageEnglish
Article numbere8133
JournalPeerJ
Volume2019
Issue number12
DOIs
Publication statusPublished - 2019

Bibliographical note

Funding Information:
This work was supported by Wellcome (105614/Z/14/Z), a Lord Kelvin Adam Smith Fellowship to Poppy H.L. Lamberton and a studentship to Lauren V. Carruthers (IBAHCM_stipend_144536) from the University of Glasgow, and the European Research Council Starting Grant (SCHISTO_PERSIST_680088). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funding Information:
The following grant information was disclosed by the authors: Wellcome: 105614/Z/14/Z. Lord Kelvin Adam Smith Fellowship to Poppy H.L. Lamberton and a Studentship to Lauren V. Carruthers: IBAHCM_stipend_144536. University of Glasgow, and the European Research Council Starting Grant: SCHISTO_PERSIST_680088.

Funding Information:
The following information was supplied relating to ethical approvals (i.e., approving body and any reference numbers): The University of Glasgow College of Medical Veterinary and Life Sciences Ethics Committee (project code: 200160068), the Vector Control Division, Ministry of Health Uganda, Research Ethics Committee (reference: VCDREC/062) and the Uganda National Council for Science and Technology (reference: UNCST-HS 2193) reviewed and approved the methods for this study.

Funding Information:
This study was approved by the University of Glasgow College of Medical Veterinary and Life Sciences Ethics Committee (project code 200160068), the Vector Control Division, Ministry of Health Uganda, Research Ethics Committee (reference: VCDREC/062) and the Uganda National Council for Science and Technology (UNCST-HS 2193). Informed signed or fingerprinted parental or guardian consent, and signed or fingerprinted assent from the study children was obtained prior to participation.

Publisher Copyright:
Copyright 2019 Carruthers et al.

ASJC Scopus subject areas

  • General Neuroscience
  • General Biochemistry,Genetics and Molecular Biology
  • General Agricultural and Biological Sciences

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