Reliability of a participant-friendly fecal collection method for microbiome analyses: a step towards large sample size investigation

Abstract

BackgroundThe effects of gut microbiota on human traits are expected to be small to moderate and adding the complexity of the human diseases, microbiome research demands big sample sizes. Fecal samples for such studies are mostly self-collected by participants at home. This imposes an extra level of complexity as sample collection and storage can be challenging. Effective, low-burden collection and storage methods allowing fecal samples to be transported properly and ensuring optimal quality and quantity of bacterial DNA for upstream analyses are necessary. Moreover, accurate assessment of the microbiome composition also depends on bacterial DNA extraction method. The aim of this study was to evaluate the reliability and efficiency of the OMNIgene•GUT kit as a participant-fecal friendly collection method (storage at room temperature for 24 h (O24h) or 7 days (O7d)) in comparison to the standard collection method (Fresh, storage at 4 °C for less than 24 h) in terms of amount of variability and information content accounting for two common DNA extraction methods.ResultsFourteen fecal samples were collected from healthy individuals (7 males, 7 females). Collection and storage methods did not differ significantly in terms of DNA concentration and Shannon diversity index. Phylum relative abundance showed significant differences for Bacteroidetes, Actinobacteria and Cyanobacteria. The differences were observed between control (Fresh) and O24h methods, but not between Fresh and O7d. These differences were not seen when performing bacterial DNA quantification based on three bacterial groups: Bacteroides spp., Bifidobacterium spp. and Clostridium cluster IV, which represent three major phyla: Bacteroidetes, Actinobacteria and Firmicutes respectively. The two DNA extraction methods differ in terms of DNA quantity, quality, bacterial diversity and bacterial relative abundance. Furthermore, principal component analysis revealed differences in microbial structure, which are driven by the DNA extraction methods more than the collection/storage methods.ConclusionOur results have highlighted the potential of using the OMNIgene•GUT kit for collection and storage at ambient temperature, which is convenient for studies aiming to collect large samples by giving participants the possibility to send samples by post. Importantly, we revealed that the choice of DNA extraction method have an impact on the microbiome profiling.Electronic supplementary materialThe online version of this article (10.1186/s12866-018-1249-x) contains supplementary material, which is available to authorized users.

Investigating the Gut Microbiota Composition of Individuals with Attention-Deficit/Hyperactivity Disorder and Association with Symptoms

“…The reason for the lack of replication between studies could be due to methodological differences. These include DNA extraction [58], 16S rRNA gene region [59], bioinformatic pipeline, data processing and analysis [60], sample size, and study design. Moreover, the age range (children vs. adults) differences between studies can underscore variation in ADHD symptoms, other non-shared environmental influences and gut microbiome composition [61,62].…”

Investigating the Gut Microbiota Composition of Individuals with Attention-Deficit/Hyperactivity Disorder and Association with Symptoms

“…The reason for the lack of replication between studies could be due to methodological differences. These include DNA extraction [55], 16S rRNA gene region [56], bioinformatic pipeline, data processing and analysis [57], sample size and study design. Moreover, the age range (children vs. adults) differences between studies can underscore variation in ADHD symptoms, other non-shared environmental influences and gut microbiome composition [58,59].…”

Impact of time and temperature on gut microbiota and SCFA composition in stool samples

“…Instead, a -20˚C freezer is available in most homes and freezing at -20˚C may also be applied PLOS ONE [38,39] but this requires willingness to place samples in the freezer and resources for shipping the samples in a frozen state. Several studies reported positive effects of preservation media and stabilizers [11][12][13][14][15][16]. However, even if patients are asked to describe how samples were handled before submission to the laboratory, it is difficult to have complete control over what has happened and samples, for diverse reasons, may have been exposed to room temperature for hours before reaching the laboratory.…”

“…A methodological problem with analyzing stool samples, however, is that the logistics of collecting samples can vary dramatically between the subjects which may influence the composition of the microbiota and its metabolic products. The current understanding is that time and temperature appears to have a low, but not negligible impact on bacterial composition and structure in stool samples [10][11][12][13][14][15]. Immediate freezing of samples at -20˚C is considered to be the "gold standard", but might not always be practically feasible.…”