Abstract
BackgroundAlterations in intestinal microbiota have been correlated with a growing number of diseases. Investigating the faecal microbiota is widely used as a non-invasive and ethically simple proxy for intestinal biopsies. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. We compared the faecal bacterial DNA quality and apparent phylogenetic composition derived using a commercial kit for stool storage and transport (DNA Genotek OMNIgene GUT) with that of freshly extracted samples, 22 from infants and 20 from older adults.ResultsUse of the storage vials increased the quality of extracted bacterial DNA by reduction of DNA shearing. When infant and elderly datasets were examined separately, no differences in microbiota composition were observed due to storage. When the two datasets were combined, there was a difference according to a Wilcoxon test in the relative proportions of Faecalibacterium, Sporobacter, Clostridium XVIII, and Clostridium XlVa after 1 week’s storage compared to immediately extracted samples. After 2 weeks’ storage, Bacteroides abundance was also significantly different, showing an apparent increase from week 1 to week 2.The microbiota composition of infant samples was more affected than that of elderly samples by storage, with significantly higher Spearman distances between paired freshly extracted and stored samples (p < 0.001). When the microbiota profiles were analysed at the operational taxonomic unit (OTU) level, three infant datasets in the study did not cluster together, while only one elderly dataset did not. The lower microbiota diversity of the infant gut microbiota compared to the elderly gut microbiota (p < 0.001) means that any alteration in the infant datasets has a proportionally larger effect.ConclusionsThe commercial storage vials appear to be suitable for high diversity microbiota samples, but may be less appropriate for lower diversity samples. Differences between fresh and stored samples mean that where storage is unavoidable, a consistent storage regime should be used. We would recommend extraction ideally within the first week of storage.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-016-0164-3) contains supplementary material, which is available to authorized users.
Highlights
Alterations in intestinal microbiota have been correlated with a growing number of diseases
In order to determine how well the OMNIgene kit preserved DNA, we compared DNA extraction under the following conditions: fresh and after both 1 and 2 weeks’ incubation at room temperature in the storage vials provided in the kit—as per instructions for use; 22 infants and 20 elderly subjects were examined under these conditions
The DNA Genotek OMNIgene gut kit offers a solution to the current common practice of patients freezing samples in home freezers, which is a barrier to participation in studies for many people
Summary
Alterations in intestinal microbiota have been correlated with a growing number of diseases. There is an urgent need for collection and transport media that would allow faecal sampling at distance from the processing laboratory, obviating the need for same-day DNA extraction recommended by previous studies of freezing and processing methods for stool. The study of the human gut microbiota is a dynamic and rapidly expanding area of research as alterations in this complex ecosystem has been correlated with a wide range of health effects on the host, including obesity [1, 2], behaviour [3] and the immune system [4], in particular autoimmune diseases [5]. Study design may not allow for immediate processing of fresh samples due to either workflow issues or geographical distance from sample collection centres to the processing laboratory. The microbiome field urgently requires a sampling method which is cost effective, applied outside of a clinical environment, and which produces an accurate representation of the microbiota composition without loss of any taxa [10]
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