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a) Relative abundance of the four main phyla for each sampling method (immediate freezing in a plain sterile tube (frozen), immediate freezing on a swab (swab frozen), using the OMNIgene•GUT kit (OMNIgene•GUT), collecting into a plain sterile tube in the post (plain post) and collecting onto a swab which is sent in the post (swab post). b) Relative Abundance of the ten main genera for each sampling method.

a) Relative abundance of the four main phyla for each sampling method (immediate freezing in a plain sterile tube (frozen), immediate freezing on a swab (swab frozen), using the OMNIgene•GUT kit (OMNIgene•GUT), collecting into a plain sterile tube in the post (plain post) and collecting onto a swab which is sent in the post (swab post). b) Relative Abundance of the ten main genera for each sampling method.

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Background Understanding the role of the gut microbiome is pivotal for the future development of therapies for the prevention and management of autoimmune conditions such as type 1 diabetes when sampling during early life may be particularly important. The current standard methods for collecting gut microbiome samples for research is to extract fre...

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... A corresponding kit for the microbiome (OMNIgene•GUT) has been used in several studies. 17,18 To our knowledge, only a few studies compared feces collection methods for simultaneous gut microbiota profiling and fecal metabolomics. 10 Notably, studies comparing collection with that of 95% EtOH and commercial fecal collection kits are lacking. ...
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Metabolites from feces provide important insights into the functionality of the gut microbiome. As immediate freezing is not always feasible in gut microbiome studies, there is a need for sampling protocols that provide the stability of the fecal metabolome and microbiome at room temperature (RT). Here, we investigated the stability of various metabolites and the microbiome (16S rRNA) in feces collected in 95% ethanol (EtOH) and commercially available sample collection kits with specific preservatives OMNImet•GUT/OMNIgene•GUT. To simulate field-collection scenarios, the samples were stored at different temperatures at varying durations (24 h + 4 °C, 24 h RT, 36 h RT, 48 h RT, and 7 days RT) and compared to aliquots immediately frozen at −80 °C. We applied several targeted and untargeted metabolomics platforms to measure lipids, polar metabolites, endocannabinoids, short-chain fatty acids (SCFAs), and bile acids (BAs). We found that SCFAs in the nonstabilized samples increased over time, while a stable profile was recorded in sample aliquots stored in 95% EtOH and OMNImet•GUT. When comparing the metabolite levels between aliquots stored at room temperature and at +4 °C, we detected several changes in microbial metabolites, including multiple BAs and SCFAs. Taken together, we found that storing samples at RT and stabilizing them in 95% EtOH yielded metabolomic results comparable to those from flash freezing. We also found that the overall composition of the microbiome did not vary significantly between different storage types. However, notable differences were observed in the α diversity. Altogether, the stability of the metabolome and microbiome in 95% EtOH provided results similar to those of the validated commercial collection kits OMNImet•GUT and OMNIgene•GUT, respectively.
... OMNIgeneGUT tubes were more practical in terms of sample collection; the correct sample volume was easy to collect, and the mixing was convenient with metal bead in the tube. OMNIgeneGUT can be convenient in field studies with at-home-collected samples and it has been used successfully in several studies (49)(50)(51). ...
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... Fecal collection and storage tubes containing 95% EtOH (OMNImet®•GUT, DNA Genotek, Canada) have been introduced as a kit tailored specifically for metabolomics analysis. A corresponding kit for the microbiome (OMNIgene®•GUT) has been used in several studies (16,17). To our knowledge, only a few studies compared feces collection methods for simultaneous gut microbiota profiling and fecal metabolomics (10). ...
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... This is an issue as exposure to ambient temperature has been shown to affect the growth or suppression of different bacterial genera within an infant stool sample. 26 This is in contrast to if a neonate is at home with a parent, who will likely know quite quickly if the neonate has defecated and thus change nappies more rapidly than hospital staff would be able. Parents could then be asked to estimate the amount of time a sample was in a nappy before collection, allowing for a measure of the exposure times to ambient temperatures of samples. ...
... Multiple studies have been conducted to evaluate the capacity of various storage methods to preserve stool microbiota. 22,[26][27][28][29][30] A recent systematic review of 92 studies found that freezing at −80°C is the most commonly used method, employed in 71.4% of studies. 23 Storage of neonatal stool samples at this temperature has been confirmed to largely preserve the microbiota for at least 2 years. ...
... Williams et al. focused on ambient temperature storage during postage of infant samples to a lab, comparing OMNIgene.Gut with dry swab and dry sterile tube storage. 26 They obtained more similar trends to Choo et al., whereby all of their storage methods showed variation from a frozen control, but the commercial buffer showed the least change and was thus deemed suitable for sample collection in study participants' homes. They also commented on the commercial kit being practical for parents to use, which is likely to maximize sampling response rates. ...
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... While comparison of swabs versus stool profiles has already been investigated on a small scale for certain defined metabolite groups (including bile acids) 10 , this has not been explored using a global profiling technique such as NMR. 1 H NMR spectroscopy detects protons within small molecules and produces a spectrum related to proton profile within the biofluid, thereby having particular utility as a global metabolic profiling technique, including host-and microbe-derived metabolites 11 . Other attractions of 1 H-NMR includes its high-throughput nature, its reproducibility, and that it is non-destructive to the samples analysed. ...
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... Additionally, rectal swabs may not obtain the same mucosal adherent microbiota as biopsy samples, which may explain the poor correlation between the sampling types 39 . Conversely, the same publications 7,38 and recent work 1,8,[40][41][42][43][44][45] have demonstrated rectal swab microbiota communities to be closely related to matched faecal samples. Although there is heterogeneity between studies in terms of sample storage, populations sampled (healthy controls or disease) and microbial analysis techniques, studies tend to demonstrate with overall consistency that rectal swabs are a reliable proxy of faecal sampling for microbiota compositional analyses. ...
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... Our analysis identified Norgen, OMNIgene-Gut, DNA/RNA Shield and PrimeStore MTM as the most efficacious stabilizers as compared to the immediately frozen aliquots (Dc). According to our results, several comparative studies have identified OMNIgene-Gut as a good DNA stabilizer for microbiome studies (Choo et al., 2015;Song et al., 2016;Abrahamson et al., 2017;Williams et al., 2019), while the other three solutions have not yet been extensively evaluated by comparative studies. In contrast, the remaining solutions tested were less efficient, showing a profile with alterations similar to unstabilized samples (S and D). ...
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... There is continuous metagenome research and development to understand the association between microbiota (e.g., inhabiting in intestinal mucosa or oral cavity) and human health. Recently, human gut health has been investigated by profiling gut microbiome compositions using fecal samples [22][23][24]. ...
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Metagenome profiling research using next-generation sequencing (NGS), a technique widely used to analyze the diversity and composition of microorganisms living in the human body, especially the gastrointestinal tract, has been actively conducted, and there is a growing interest in the quantitative and diagnostic technology for specific microorganisms. According to recent trends, quantitative real-time PCR (qRT-PCR) is still a considerable technique in detecting and quantifying bacteria associated with the human oral and nasal cavities, due to the analytical cost and time burden of NGS technology. Here, based on NGS metagenome profiling data produced by utilizing 100 gut microbiota samples, we conducted a comparative analysis for the identification and quantification of five bacterial genera (Akkermansia, Bacteroides, Bifidobacterium, Phascolarctobacterium, and Roseburia) within same metagenomic DNA samples through qRT-PCR assay in parallel. Genus-specific primers, targeting the particular gene of each genus for qRT-PCR assay, allowed a statistically consistent quantification pattern with the metagenome profiling data. Furthermore, results of bacterial identification through Sanger validation demonstrated the high genus-specificity of each primer set. Therefore, our study suggests that an approach to quantifying specific microorganisms by applying the qRT-PCR method can compensate for the concerns (potential issues) of NGS while also providing efficient benefits to various microbial industries.
... These developments of continuous metagenome research have led to efforts to establish an association between microbiota (e.g., inhabiting in intestinal mucosa or oral cavity) and human health (Mohajeri et al. 2018;Young 2017). Recently, human gut health has been checked by pro ling the gut microbiome composition through feces samples (Abrahamson et al. 2017;Liang et al. 2020;Williams et al. 2019). ...
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Metagenome profiling research using next-generation sequencing (NGS), a technique widely used to analyze the diversity and composition of microorganisms living in the human body, especially the gastrointestinal tract, has been activated, and there is a growing interest in the quantitative and diagnostic technology for specific microorganisms. According to recent trends, quantitative real-time PCR (qRT-PCR) are still of considerable technique in detecting and quantifying bacteria associated with the human mouth, nasal cavity, and pharynx due to analytical cost and time burden of NGS technology. Here, based on NGS metagenome profiling data produced by utilizing 100 gut microbiota samples, we conducted a comparative analysis of identifying for five bacterial genera proportions ( Akkermansia , Bacteroides , Bifidobacterium , Phascolarctobacterium , and Roseburia ) within same metagenomic DNA samples through qRT-PCR assay in parallel. Genus-specific primer, targeting the particular gene of each genus for qRT-PCR assay, allowed a statistically consistent quantification pattern with the metagenome profiling data. Furthermore, results of bacterial identification through Sanger validation demonstrated the high genus-specificity of each primer set. Therefore, our study suggests that an approach to quantifying specific microorganisms by applying qRT-PCR method can compensate for the concerns (potential issues) of NGS while also providing efficient benefits to various microbial industries
... Understanding on gut microbiome has increased tremendously due to technological advancements such as molecular sequencing and big data analysis (Azad et al. 2018). Microbial diversity in fecal samples is a direct reflection of gut microbiome as reported by several research groups (Seekatz et al. 2014;Ezzy et al. 2019;Williams et al. 2019). Imbalance in gut microbial communities, termed as 'dysbiosis', contributes significantly to the pathophysiology of IBS (Menees and Chey 2018). ...
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Bacillus coagulans LBSC showed stability in acidic pH, bile and simulated human gastrointenstinal juices. Under static gut model, when passed through oral, gastric and intestinal phases, B. coagulans LBSC was found to be stable as free viable spores and also with various foods such as milk and baby foods, as well as American and European diets. In human studies, modulation of gut microbiota by B. coagulans LBSC was comprehended by whole genome metagenome analysis of fecal samples obtained from pre- and post-treatment of irritable bowel syndrome (IBS) patients. B. coagulans LBSC treatment showed positive modulation in gut microbiota, especially up regulation of phyla such as Actinobacteria and Firmicutes, whereas down regulation of Bacteroids, Proteobacteria, Streptophyta and Verrucomicrobia. Simultaneously, it has altered various microbiota associated metabolic pathways to create the normalcy of gut microenvironment.
... During the second visit (2-3 days after visit 1), participants were provided with a stool sample kit (OMNIgene® Gut kit) and were asked to follow directions to collect a stool sample prior their third visit (3-5 days after visit 2). OMNIgene Gut kits keep samples stable at ambient temperature for up to 60 days [65,66]. Sample tubes were frozen at -80 degrees immediately upon receipt, and later sent The read pairs were demultiplexed based on unique molecular barcodes added via PCR during library generation, then merged using USEARCH v7.0.1090 [70]. ...
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Background: Traditional thinking is that physical activity benefits mental and physical health, however, excessive physical activity can increase anxiety, depression, and affect the gut microbiome. Considering the strong connection between the gut and the brain, the purpose of the present study was to evaluate the association between gut microbiota composition and anxiety as well as depression in highly active individuals. Methods: Participants included 55 young adults (ages 18-25, 51% males). All participants were highly physically active, as determined by 7 days of SenseWear monitoring. Anxiety and depression were measured with the Beck Anxiety and Depression Inventories. Alpha diversity, beta diversity, and microbial composition were evaluated via 16S rRNA gene sequencing using distal gut samples. Results: Greater anxiety was associated with both lower distal gut alpha diversity ( P < 0.05) and higher beta diversity (PERMANOVA test; R-squared: 0.17562, P = 0.027), which appeared stronger in males. Genus level taxonomic abundance analysis showed Prevotella relative abundance as higher in males with higher anxiety ( P = 0.03, q=0.06). However, adjusted linear regression analysis, controlling for fiber intake and sex nullified the association between Prevotella and anxiety. Additional analysis demonstrated a strong association between lower dietary fiber intake and higher anxiety scores (Est.= -0.48, SE= 0.20 , P = 0.021). Conclusion: In highly active individuals, specifically males, there is a strong relationship between the gut microbiome, fiber intake, and anxiety. These data suggest highly active males with anxiety may benefit from increased dietary fiber intake.