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Human faecal collection methods demonstrate a bias in microbiome composition by cell wall structure.

ABSTRACT: Clinical trial faecal collections present challenges through geographical spread and inexperienced participants. Collection techniques have been developed and tested to overcome these challenges, but previous studies investigating these techniques have demonstrated a highly variable capacity for sample preservation. Furthermore, these studies typically only examine either preservation of genetic content or metabolites, not both. This study investigated the Stool Nucleic Acid Collection and Preservation Tube (Norgen BioTek Corp) for the preservation of both microbial DNA and microbial organic acid metabolites in human faecal samples when compared to frozen samples. Twenty six healthy adult participants were instructed to collect a bowel movement, subsample into collection tubes and immediately transfer the remaining bulk to -20 °C storage. Resulting organic acid concentrations remained comparable across methods when the preservation tubes were used correctly. The 16S rRNA gene sequencing data revealed twenty significantly different bacterial genera between the two collection methods. Ten Gram-negative genera were more abundant in the collection tubes, and ten Gram-positive genera were more abundant in the fresh frozen samples. This study has illustrated that faecal collection methods bias the microbial community profile according to Gram status and this should be considered when designing studies that collect and store human faecal samples.

SUBMITTER: Watson EJ

PROVIDER: S-EPMC6856092 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMicrobiota profiles are strongly influenced by many technical aspects that impact the ability of researchers to compare results. To investigate and identify potential biases introduced by technical variations, we compared several approaches throughout the entire workflow of a microbiome study, from sample collection to sequencing, using commercially available mock communities (from bacterial strains as well as from DNA) and multiple human fecal samples, including a large set of positive controls created as a random mix of several participant samples.MethodsHuman fecal material was sampled, and aliquots were used to test two commercially available stabilization solutions (OMNIgene·GUT and Zymo Research) in comparison to samples frozen immediately upon collection. In addition, the methodology for DNA extraction, input of DNA, or the number of PCR cycles were analyzed. Furthermore, to investigate the potential batch effects in DNA extraction, sequencing, and barcoding, we included 139 positive controls.ResultsSamples preserved in both the stabilization buffers limited the overgrowth of Enterobacteriaceae when compared to unpreserved samples stored at room temperature (RT). These stabilized samples stored at RT were different from immediately frozen samples, where the relative abundance of Bacteroidota was higher and Actinobacteriota and Firmicutes were lower. As reported previously, the method used for cell disruption was a major contributor to variation in microbiota composition. In addition, a high number of cycles during PCR lead to an increase in contaminants detected in the negative controls. The DNA extraction had a significant impact on the microbial composition, also observed with the use of different Illumina barcodes during library preparation and sequencing, while no batch effect was observed in replicate runs.ConclusionOur study reaffirms the importance of the mechanical cell disruption method and immediate frozen storage as critical aspects in fecal microbiota studies. A comparison of storage conditions revealed that the bias was limited in RT samples preserved in stabilization systems, and these may be a suitable compromise when logistics are challenging due to the size or location of a study. Moreover, to reduce the effect of contaminants in fecal microbiota profiling studies, we suggest the use of ~125 pg input DNA and 25 PCR cycles as optimal parameters during library preparation.

Project description:Background: Integrated microbiome and metabolomics analyses hold the potential to reveal interactions between host and microbiota in relation to disease risks. However, there are few studies evaluating how field methods influence fecal microbiome characterization and metabolomics profiling. Methods: Five fecal collection methods [immediate freezing at -20°C without preservative, OMNIgene GUT, 95% ethanol, RNAlater, and Flinders Technology Associates (FTA) cards] were used to collect 40 fecal samples from eight healthy volunteers. We performed gut microbiota 16S rRNA sequencing, untargeted metabolomics profiling, and targeted metabolomics focusing on short chained fatty acids (SCFAs). Metrics included ?-diversity and ?-diversity as well as distributions of predominant phyla. To evaluate the concordance with the "gold standard" immediate freezing, the intraclass correlation coefficients (ICCs) for alternate fecal collection systems were calculated. Correlations between SCFAs and gut microbiota were also examined. Results: The FTA cards had the highest ICCs compared to the immediate freezing method for ?-diversity indices (ICCs = 0.96, 0.96, 0.76 for Shannon index, Simpson's Index, Chao-1 Index, respectively), followed by OMNIgene GUT, RNAlater, and 95% ethanol. High ICCs (all >0.88) were observed for all methods for the ?-diversity metric. For untargeted metabolomics, in comparison to immediate freezing which detected 621 metabolites at ?75% detectability level, 95% ethanol showed the largest overlapping set of metabolites (n = 430; 69.2%), followed by FTA cards (n = 330; 53.1%) and OMNIgene GUT (n = 213; 34.3%). Both OMNIgene GUT (ICCs = 0.82, 0.93, 0.64) and FTA cards (ICCs = 0.87, 0.85, 0.54) had acceptable ICCs for the top three predominant SCFAs (butyric acid, propionic acid and acetic acid). Nominally significant correlations between bacterial genera and SCFAs (P < 0.05) were observed in fecal samples collected by different methods. Of note, a high correlation between the genus Blautia (known butyrate producer) and butyric acid was observed for both immediate freezing (r = 0.83) and FTA cards (r = 0.74). Conclusions: Four alternative fecal collection methods are generally comparable with immediate freezing, but there are differences in certain measures of the gut microbiome and fecal metabolome across methods. Choice of method depends on the research interests, simplicity of fecal collection procedures and ease of transportation to the lab, especially for large epidemiological studies.

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Human faecal collection methods demonstrate a bias in microbiome composition by cell wall structure.

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