Influence of fecal collection conditions and 16S rRNA gene sequencing at two centers on human gut microbiota analysis

Simon C Barry,Jennifer Couper ,Jocelyn Sietsma Penington,Alexandra J Roth‐Schulze ,Megan A S Penno,Leonard C Harrison,Katrina Ngui ,Cheryl Y Brown,Stephen Wilcox ,Nadim J Ajami,John M Wentworth,Anthony T Papenfuss,Esther Bandala‐Sanchez ,Joseph F Petrosino

doi:10.1038/s41598-018-22491-7

Abstract

To optimise fecal sampling for reproducible analysis of the gut microbiome, we compared different methods of sample collection and sequencing of 16S rRNA genes at two centers. Samples collected from six individuals on three consecutive days were placed in commercial collection tubes (OMNIgeneGut OMR-200) or in sterile screw-top tubes in a home fridge or home freezer for 6–24 h, before transfer and storage at −80 °C. Replicate samples were shipped to centers in Australia and the USA for DNA extraction and sequencing by their respective PCR protocols, and analysed with the same bioinformatic pipeline. Variation in gut microbiome was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection, and between the two centers. We conclude that collection with storage and transport at 4 °C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. Other factors including differences in PCR and sequencing methods account for relatively minor variation compared to differences between individuals.

Highlights

The fecal or ‘gut’ microbiome is shaped strongly by diet and by the host genotype, age, hygiene and antibiotic exposure, and is altered in many pathophysiological states[1,2]
Samples immediately collected into these tubes and stored for three days at room temperature showed little difference in microbial composition by 16S rRNA gene sequencing compared with samples immediately frozen at −80 °C7
As microbiome studies expand into larger populations at multiple sites, stringent quality control remains critical. With this in mind and to optimise analysis of the gut microbiome in a multi-site, longitudinal pregnancy-birth cohort study[16], we evaluated the impact of different collection-processing methods on three sequential daily fecal samples from six individuals for 16S rRNA gene sequencing in two centers

Summary

Introduction

The fecal or ‘gut’ microbiome is shaped strongly by diet and by the host genotype, age, hygiene and antibiotic exposure, and is altered in many pathophysiological states[1,2]. Previous studies have shown that fecal microbial composition overall was not altered when DNA was extracted from a fresh fecal sample compared to a sample that had been immediately frozen and stored at −80 °C for up to 6 months[4,5]. 200) collection and liquid storage tube, which is reported to stabilize DNA at room temperature for 14 days[13]. Samples immediately collected into these tubes and stored for three days at room temperature showed little difference in microbial composition by 16S rRNA gene sequencing compared with samples immediately frozen at −80 °C7. A similar result was obtained when samples in OMR-200tubes stored for 1–28 days at room temperature www.nature.com/scientificreports/. The relative abundance of Bacteroides increased after seven days and in infants (who had lower microbial diversity than adults) significant differences from fresh samples were observed after 14 days storage

Methods

Results

Conclusion