Computational protocol: Allergy associations with the adult fecal microbiota: Analysis of the American Gut Project

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Protocol publication

[…] The 16S rRNA V4 region was sequenced by the American Gut Project (AGP). The operational taxonomic unit (OTU) table rarefied to 10,000 sequence reads per sample, as well as metadata, was downloaded from the AGP website ( Samples with less than 10,000 sequence reads were excluded from analysis. A current summary is available at, and details of the OTU picking and taxonomy assignment are available at Richness (number of observed species), alpha diversity metrics [Shannon index, Chao1, phylogenetic diversity (PD)_whole_tree], beta diversity metrics (weighted and unweighted UniFrac distance matrices), and relative abundance of each taxon were calculated in the Quantitative Insights Into Microbial Ecology (QIIME) pipeline ().After exclusions [duplicates, diabetes, inflammatory bowel disease, age < 4 years (after which the microbiota resembles that of adults ()), missing race, specimen not feces, antibiotic used in the past month], data were analyzed for 1879 AGP participants. Each participant who provided a positive response on the AGP self-administered questionnaire was classified as having an allergy or pet. For foods, the verbatim question, which did not require validation by a physician, was: “I am allergic to ___ (mark all that apply): Peanuts, Tree nuts, Shellfish, Other, I have no food allergies that I know of.” For non-foods, there were three verbatim questions: “Do you have any of the following non-food allergies? Mark all that apply: Drug (e.g. Penicillin), Pet dander, Beestings, Poison ivy/oak”; “Do you have seasonal allergies? Yes/No”; and Have you been diagnosed with any of the following conditions (check all which apply)? … (e) Asthma, Cystic Fibrosis or Lung Disease.… (v) Skin Condition….” Thus, the allergies included four foods (peanuts, tree nuts, shellfish, other food) and six non-foods [drug, bee sting, dander, asthma, seasonal, and eczema (specified in skin conditions)]. For pets, the questions were: “Do you have a dog?” and “Do you have a cat?” Participants with an affirmative response were compared to participants without an affirmative response. In sensitivity analyses (specifically, dander allergy with dog or cat ownership in Supplemental Online Content), excluding participants with uncertain or no response reduced sample size and statistical power but had no substantive effect on the associations. We previously noted that AGP participants are widely scattered across the US and resemble the American adult population with respect to the prevalence of cesarean birth and appendectomy, but they are overwhelmingly non-Hispanic Caucasian (93%) and non-smokers (96%) (). In like manner for the current analysis, we compared the prevalence of allergies reported in AGP data to the prevalence of clinical allergens that were self-reported in representative samples of the US population, particularly the National Health and Nutrition Examination Survey 2005–2006 (, , , ). [...] Weighted and unweighted UniFrac distance matrices were derived from the QIIME pipeline. For each allergy trait, we used the Microbiome Regression-based Kernel Association Test (MiRKAT) (), a kernel-based regression method, for testing whether microbiome composition differed between cases and controls using either the weighted or unweighted UniFrac distance matrix. The associations were adjusted for sex, age, BMI, season, time since last antibiotic use, probiotic and vitamin use. For each significant association, we used MiRKAT to run 100,000 permutations to verify the asymptotic P-value approximations. We identified significant associations by controlling false discovery rate (FDR) < 10%. We also performed principal coordinate analysis (PCoA) to derive the top three PCoA scores and examined their associations with allergy traits. […]

Pipeline specifications

Software tools QIIME, UniFrac, MiRKAT
Applications Phylogenetics, 16S rRNA-seq analysis
Diseases Asthma, Eczema, Food Hypersensitivity, Hypersensitivity, Peanut Hypersensitivity