Technical variation in metagenomic analysis must be minimized to confidently assess the contributions of microbiota to human health. Here we tested 21 representative DNA extraction protocols on the same fecal samples and quantified differences in observed microbial community composition. We compared them with differences due to library preparation and sample storage, which we contrasted with observed biological variation within the same specimen or within an individual over time. We found that DNA extraction had the largest effect on the outcome of metagenomic analysis. To rank DNA extraction protocols, we considered resulting DNA quantity and quality, and we ascertained biases in estimates of community diversity and the ratio between Gram-positive and Gram-negative bacteria. We recommend a standardized DNA extraction method for human fecal samples, for which transferability across labs was established and which was further benchmarked using a mock community of known composition. Its adoption will improve comparability of human gut microbiome studies and facilitate meta-analyses.

SUMMARY Background A low fermentable oligosaccharides, disaccharides, monosaccharides, and polyols (FODMAP) diet can ameliorate symptoms in adult irritable bowel syndrome (IBS) within 48 hours. Aim To determine the efficacy of a low FODMAP diet in childhood IBS and whether gut microbial composition and/or metabolic capacity are associated with its efficacy. Methods In a double-blind, crossover trial, children with Rome III IBS completed a one-week baseline period. They then were randomized to a low FODMAP diet or typical American childhood diet (TACD), followed by a 5-day washout period before crossing over to the other diet. GI symptoms were assessed with abdominal pain frequency being the primary outcome. Baseline gut microbial composition (16S rRNA sequencing) and metabolic capacity (PICRUSt) were determined. Metagenomic biomarker discovery (LEfSe) compared Responders (≥50% decrease in abdominal pain frequency on low FODMAP diet only) versus Non-Responders (no improvement during either intervention). Results Thirty-three children completed the study. Less abdominal pain occurred during the low FODMAP diet versus TACD (1.1 ± 0.2 (SEM) episodes/day versus 1.7 ± 0.4, P<0.05). Compared to baseline (1.4 ± 0.2), children had fewer daily abdominal pain episodes during the low FODMAP diet (P<0.01) but more episodes during the TACD (P<0.01). Responders were enriched at baseline in taxa with known greater saccharolytic metabolic capacity (e.g., Bacteroides, Ruminococcaceae, Faecalibacterium prausnitzii) and 3 KEGG orthologs, of which two relate to carbohydrate metabolism. Conclusions In childhood IBS, a low FODMAP diet decreases abdominal pain frequency. Gut microbiome biomarkers may be associated with low FODMAP diet efficacy.

BackgroundThe gut microbiome influences myriad host functions, including nutrient acquisition, immune modulation, brain development, and behavior. Although human gut microbiota are recognized to change as we age, information regarding the structure and function of the gut microbiome during childhood is limited. Using 16S rRNA gene and shotgun metagenomic sequencing, we characterized the structure, function, and variation of the healthy pediatric gut microbiome in a cohort of school-aged, pre-adolescent children (ages 7–12 years). We compared the healthy pediatric gut microbiome with that of healthy adults previously recruited from the same region (Houston, TX, USA).ResultsAlthough healthy children and adults harbored similar numbers of taxa and functional genes, their composition and functional potential differed significantly. Children were enriched in Bifidobacterium spp., Faecalibacterium spp., and members of the Lachnospiraceae, while adults harbored greater abundances of Bacteroides spp. From a functional perspective, significant differences were detected with respect to the relative abundances of genes involved in vitamin synthesis, amino acid degradation, oxidative phosphorylation, and triggering mucosal inflammation. Children’s gut communities were enriched in functions which may support ongoing development, while adult communities were enriched in functions associated with inflammation, obesity, and increased risk of adiposity.ConclusionsPrevious studies suggest that the human gut microbiome is relatively stable and adult-like after the first 1 to 3 years of life. Our results suggest that the healthy pediatric gut microbiome harbors compositional and functional qualities that differ from those of healthy adults and that the gut microbiome may undergo a more prolonged development than previously suspected.Electronic supplementary materialThe online version of this article (doi:10.1186/s40168-015-0101-x) contains supplementary material, which is available to authorized users.