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

Background To optimise fecal sampling and analysis yielding reproducible microbiome data, and gain further insight into sources of its variation, we compared different collection conditions and 16S rRNA gene sequencing protocols in two centers. Fecal samples were collected on three sequential days from six healthy adults and placed in commercial collection tubes (OMNIgeneGut OMR-200) at room temperature or in sterile 5 ml screw-top tubes in a home fridge or home freezer for 6-24 h, before transfer at 4°C to the laboratory and storage at - 80°C within 24 hours. Replicate samples were shipped on dry ice to centers in Australia and the USA for DNA extraction and sequencing of the V4 region of the 16S rRNA gene, using different PCR protocols. Sequences were analysed with the QIIME pipeline and Greengenes database at the Australian center and with an in-house pipeline and SILVA database at the USA center. Results Variation in gut microbiome composition and diversity was dominated by differences between individuals. Minor differences in the abundance of taxa were found between collection-processing methods and day of collection. Larger differences were evident between the two centers, including in the relative abundances of genus Akkermansia , in phylum Verrucomicrobiales , and Bifidobacteria in Actinobacteria . Conclusions Collection with storage and transport at 4°C within 24 h is adequate for 16S rRNA analysis of the gut microbiome. However, variation between sequencing centers suggests that cohort samples should be sequenced by the same method in one center. Differences in handling, shipping and methods of PCR gene amplification and sequence analysis in different centers introduce variation in ways that are not fully understood. These findings are particularly relevant as microbiome studies shift towards larger population-based and multicenter studies. * ENDIA : Environmental determinants of islet autoimmunity BCM : Baylor College of Medicine WEHI : Walter and Eliza Hall Institute of Medical Research 16Sv4 : hypervariable region of the 16S rRNA marker gene OTU : Operational Taxonomic Unit NMDS : Non-metric Multi-Dimensional Scaling WW : WEHI bioinformatics analysis applied to DNA sequenced at WEHI WB : WEHI bioinformatics analysis applied to DNA sequenced at BCM BB : BCM bioinformatics analysis applied to DNA sequenced at BCM (supplementary material)