The trans-ancestral genomic architecture of glycemic traits.

Glycemic traits are used to diagnose and monitor type 2 diabetes and cardiometabolic health. To date, most genetic studies of glycemic traits have focused on individuals of European ancestry. Here we aggregated genome-wide association studies comprising up to 281,416 individuals without diabetes (30% non-European ancestry) for whom fasting glucose, 2-h glucose after an oral glucose challenge, glycated hemoglobin and fasting insulin data were available. Trans-ancestry and single-ancestry meta-analyses identified 242 loci (99 novel; P < 5 × 10-8), 80% of which had no significant evidence of between-ancestry heterogeneity. Analyses restricted to individuals of European ancestry with equivalent sample size would have led to 24 fewer new loci. Compared with single-ancestry analyses, equivalent-sized trans-ancestry fine-mapping reduced the number of estimated variants in 99% credible sets by a median of 37.5%. Genomic-feature, gene-expression and gene-set analyses revealed distinct biological signatures for each trait, highlighting different underlying biological pathways. Our results increase our understanding of diabetes pathophysiology by using trans-ancestry studies for improved power and resolution.

Distinct immune phenotypes in infants developing asthma during childhood.

Early exposure to environmental triggers may elicit trajectories to chronic inflammatory disease through deregulated immune responses. To address relations between early immune competence and development of childhood asthma, we performed functional immune profiling of 186 parameters in blood of 541 18-month-old infants and examined links between their response phenotype and development of transient or persistent disease at 6 years of age. An abnormal neutrophil-linked antiviral response was associated with increased risk of transient asthma. Children who exhibited persistent asthma at year 6 showed enhanced interleukin-5 (IL-5) and IL-13 production in stimulated T cells at 18 months of age, which was associated with early life bacterial colonization of the airways. These findings highlight the early appearance of distinct immune characteristics in infants developing different asthma endotypes during childhood.

Infant airway microbiota and topical immune perturbations in the origins of childhood asthma.

Asthma is believed to arise through early life aberrant immune development in response to environmental exposures that may influence the airway microbiota. Here, we examine the airway microbiota during the first three months of life by 16S rRNA gene amplicon sequencing in the population-based Copenhagen Prospective Studies on Asthma in Childhood 2010 (COPSAC2010) cohort consisting of 700 children monitored for the development of asthma since birth. Microbial diversity and the relative abundances of Veillonella and Prevotella in the airways at age one month are associated with asthma by age 6 years, both individually and with additional taxa in a multivariable model. Higher relative abundance of these bacteria is furthermore associated with an airway immune profile dominated by reduced TNF-α and IL-1ß and increased CCL2 and CCL17, which itself is an independent predictor for asthma. These findings suggest a mechanism of microbiota-immune interactions in early infancy that predisposes to childhood asthma.

Genetic, Clinical, and Environmental Factors Associated With Persistent Atopic Dermatitis in Childhood.

Importance: Knowledge about factors associated with persistence of atopic dermatitis (AD) during childhood is sparse. Objective: To explore heritable, environmental, and clinical factors associated with persistent AD based on 13 years' follow-up of an at-risk birth cohort. Design, Setting, and Participants: In the Copenhagen Prospective Study on Asthma in Childhood 2000 (COPSAC2000) clinical birth cohort study, 411 children born to mothers with asthma were followed up until the age of 13 years at a clinical research unit in Copenhagen, Denmark, from August 1998 to June 2015. Atopic dermatitis was diagnosed prospectively during close clinical follow-up according to the criteria of Hanifin and Rajka. Data were gathered on parental history, social circumstances, and environmental factors through parent interviews. The cohort was followed up with biannual visits to the clinic until the age of 7 years and were seen again at age 13 years. Data were analyzed from August 2015 to January 2018. Main Outcomes and Measures: Atopic dermatitis was diagnosed using Hanifin and Rajka major and minor criteria, and severity was determined by Scoring Atopic Dermatitis (SCORAD) index, with possible scores from 0 to 83, with higher scores indicating more severe AD. Results: Of the 411 children in the cohort, 203 (49.4%) were male and 186 (45.3%) were diagnosed with AD before the age of 13 years; 40 of 166 children (24.1%) had persistent AD at the age of 13 years, and 126 (76.0%) experienced remission. Factors associated with persistent AD to age 13 years included heritability, environmental exposures, asthma and allergic sensitization, clinical presentation at the time of diagnosis, the composition of Hanifin and Rajka diagnostic minor criteria, and AD severity according to SCORAD. A higher AD genetic risk score was associated with an increased the risk for persistent AD (multivariable odds ratio [OR], 1.8; 95% CI, 1.1-2.9; P = .02), together with paternal asthma (multivariable OR, 3.7; 95% CI, 1.2-11.5; P = .02); paternal AD (multivariable OR, 6.2; 95% CI, 1.17-23.2; P = .007), and higher social circumstances (multivariable OR, 1.6; 95% CI, 1.0-2.5; P = .05). Particular clinical presentations at time of diagnosis were also associated with specific minor criteria of Hanifin and Rajka (Dennie-Morgan and anterior neck folds, white dermographism, intolerance to wool, itching when sweating, tendency to skin infection, food intolerance, and food allergy) (OR, 2.6; 95% CI, 1.1-6.2; P = .03) as well as increased severity at diagnosis (OR, 1.1; 95% CI, 1.0-1.1; P = .007). Conclusions and Relevance: In a birth cohort of children at risk for asthma who received close clinical follow-up to age 13 years, known genetic AD risk variants, paternal asthma and AD, high social circumstances, diagnostic minor criteria, and disease severity at onset were associated with persistent AD at age 13 years. These findings may be applied in clinical practice to evaluate the likely disease course for individual patients.

A novel rare CUBN variant and three additional genes identified in Europeans with and without diabetes: results from an exome-wide association study of albuminuria.

AIMS/HYPOTHESIS: Identifying rare coding variants associated with albuminuria may open new avenues for preventing chronic kidney disease and end-stage renal disease, which are highly prevalent in individuals with diabetes. Efforts to identify genetic susceptibility variants for albuminuria have so far been limited, with the majority of studies focusing on common variants. METHODS: We performed an exome-wide association study to identify coding variants in a two-stage (discovery and replication) approach. Data from 33,985 individuals of European ancestry (15,872 with and 18,113 without diabetes) and 2605 Greenlanders were included. RESULTS: We identified a rare (minor allele frequency [MAF]: 0.8%) missense (A1690V) variant in CUBN (rs141640975, ß = 0.27, p = 1.3 × 10-11) associated with albuminuria as a continuous measure in the combined European meta-analysis. The presence of each rare allele of the variant was associated with a 6.4% increase in albuminuria. The rare CUBN variant had an effect that was three times stronger in individuals with type 2 diabetes compared with those without (pinteraction = 7.0 × 10-4, ß with diabetes = 0.69, ß without diabetes = 0.20) in the discovery meta-analysis. Gene-aggregate tests based on rare and common variants identified three additional genes associated with albuminuria (HES1, CDC73 and GRM5) after multiple testing correction (pBonferroni < 2.7 × 10-6). CONCLUSIONS/INTERPRETATION: The current study identifies a rare coding variant in the CUBN locus and other potential genes associated with albuminuria in individuals with and without diabetes. These genes have been implicated in renal and cardiovascular dysfunction. The findings provide new insights into the genetic architecture of albuminuria and highlight target genes and pathways for the prevention of diabetes-related kidney disease.

Author Correction: Genome-wide association and HLA fine-mapping studies identify risk loci and genetic pathways underlying allergic rhinitis.

In the version of this article initially published, in Fig. 3, the y-axis numbering did not match the log scale indicated in the axis label. The error has been corrected in the HTML and PDF version of the article.

NKG2D gene variation and susceptibility to viral bronchiolitis in childhood.

BACKGROUND: Genetic factors associated with bronchiolitis are inadequately characterized. We therefore inspected a selected subpopulation of our previous genome-wide association study (GWAS) of bronchiolitis for overlap with known quantitative trait loci (QTLs) to identify susceptibility loci that potentially affect mRNA and protein levels. METHODS: GWAS included a Finnish-Swedish case-control population (n = 187), matched for age and site. We integrated GWAS variants (p < 10-4) with QTL data. We subsequently verified allele-specific expression of identified QTLs by flow cytometry. Association of the resulting candidate loci with bronchiolitis was tested in three additional cohorts from Finland and Denmark (n = 1201). RESULTS: Bronchiolitis-susceptibility variant rs10772271 resided within QTLs previously associated with NKG2D (NK group 2, member D) mRNA and protein levels. Flow cytometric analysis confirmed the association with protein level in NK cells. The GWAS susceptibility allele (A) of rs10772271 (odds ratio [OR] = 2.34) corresponded with decreased NKG2D expression. The allele was nominally associated with bronchiolitis in one Finnish replicate (OR = 1.50), and the other showed directional consistency (OR = 1.43). No association was detected in Danish population CONCLUSIONS: The bronchiolitis GWAS susceptibility allele was linked to decreased NKG2D expression in the QTL data and in our expression analysis. We propose that reduced NKG2D expression predisposes infants to severe bronchiolitis.

Genome-wide association and HLA fine-mapping studies identify risk loci and genetic pathways underlying allergic rhinitis.

Allergic rhinitis is the most common clinical presentation of allergy, affecting 400 million people worldwide, with increasing incidence in westernized countries1,2. To elucidate the genetic architecture and understand the underlying disease mechanisms, we carried out a meta-analysis of allergic rhinitis in 59,762 cases and 152,358 controls of European ancestry and identified a total of 41 risk loci for allergic rhinitis, including 20 loci not previously associated with allergic rhinitis, which were confirmed in a replication phase of 60,720 cases and 618,527 controls. Functional annotation implicated genes involved in various immune pathways, and fine mapping of the HLA region suggested amino acid variants important for antigen binding. We further performed genome-wide association study (GWAS) analyses of allergic sensitization against inhalant allergens and nonallergic rhinitis, which suggested shared genetic mechanisms across rhinitis-related traits. Future studies of the identified loci and genes might identify novel targets for treatment and prevention of allergic rhinitis.

Consortium-based genome-wide meta-analysis for childhood dental caries traits.

Prior studies suggest dental caries traits in children and adolescents are partially heritable, but there has been no large-scale consortium genome-wide association study (GWAS) to date. We therefore performed GWAS for caries in participants aged 2.5-18.0 years from nine contributing centres. Phenotype definitions were created for the presence or absence of treated or untreated caries, stratified by primary and permanent dentition. All studies tested for association between caries and genotype dosage and the results were combined using fixed-effects meta-analysis. Analysis included up to 19 003 individuals (7530 affected) for primary teeth and 13 353 individuals (5875 affected) for permanent teeth. Evidence for association with caries status was observed at rs1594318-C for primary teeth [intronic within ALLC, odds ratio (OR) 0.85, effect allele frequency (EAF) 0.60, P 4.13e-8] and rs7738851-A (intronic within NEDD9, OR 1.28, EAF 0.85, P 1.63e-8) for permanent teeth. Consortium-wide estimated heritability of caries was low [h2 of 1% (95% CI: 0%: 7%) and 6% (95% CI 0%: 13%) for primary and permanent dentitions, respectively] compared with corresponding within-study estimates [h2 of 28% (95% CI: 9%: 48%) and 17% (95% CI: 2%: 31%)] or previously published estimates. This study was designed to identify common genetic variants with modest effects which are consistent across different populations. We found few single variants associated with caries status under these assumptions. Phenotypic heterogeneity between cohorts and limited statistical power will have contributed; these findings could also reflect complexity not captured by our study design, such as genetic effects which are conditional on environmental exposure.

Enhancer and Transcription Factor Dynamics during Myeloid Differentiation Reveal an Early Differentiation Block in Cebpa null Progenitors.

Transcription factors PU.1 and CEBPA are required for the proper coordination of enhancer activity during granulocytic-monocytic (GM) lineage differentiation to form myeloid cells. However, precisely how these factors control the chronology of enhancer establishment during differentiation is not known. Through integrated analyses of enhancer dynamics, transcription factor binding, and proximal gene expression during successive stages of murine GM-lineage differentiation, we unravel the distinct kinetics by which PU.1 and CEBPA coordinate GM enhancer activity. We find no evidence of a pioneering function of PU.1 during late GM-lineage differentiation. Instead, we delineate a set of enhancers that gain accessibility in a CEBPA-dependent manner, suggesting a pioneering function of CEBPA. Analyses of Cebpa null bone marrow demonstrate that CEBPA controls PU.1 levels and, unexpectedly, that the loss of CEBPA results in an early differentiation block. Taken together, our data provide insights into how PU.1 and CEBPA functionally interact to drive GM-lineage differentiation.

Publisher Correction: Maturation of the gut microbiome and risk of asthma in childhood.

The originally published version of this Article contained an incorrect version of Figure 3 that was introduced following peer review and inadvertently not corrected during the production process. Both versions contain the same set of abundance data, but the incorrect version has the children's asthma status erroneously disconnected from the abundance data, thereby producing the non-representative p values and graphic presentations. These errors have now been rectified, with the correct version of Figure 3 replaced in both the PDF and HTML versions of the Article.

Maturation of the gut microbiome and risk of asthma in childhood.

The composition of the human gut microbiome matures within the first years of life. It has been hypothesized that microbial compositions in this period can cause immune dysregulations and potentially cause asthma. Here we show, by associating gut microbial composition from 16S rRNA gene amplicon sequencing during the first year of life with subsequent risk of asthma in 690 participants, that 1-year-old children with an immature microbial composition have an increased risk of asthma at age 5 years. This association is only apparent among children born to asthmatic mothers, suggesting that lacking microbial stimulation during the first year of life can trigger their inherited asthma risk. Conversely, adequate maturation of the gut microbiome in this period may protect these pre-disposed children.

Cadherin-related Family Member 3 Genetics and Rhinovirus C Respiratory Illnesses.

RATIONALE: Experimental evidence suggests that CDHR3 (cadherin-related family member 3) is a receptor for rhinovirus (RV)-C, and a missense variant in this gene (rs6967330) is associated with childhood asthma with severe exacerbations. OBJECTIVES: To determine whether rs6967330 influences RV-C infections and illnesses in early childhood. METHODS: We studied associations between rs6967330 and respiratory infections and illnesses in the COPSAC2010 (Copenhagen Prospective Studies on Asthma in Childhood 2010) and COAST (Childhood Origins of Asthma Birth Cohort Study) birth cohorts, where respiratory infections were monitored prospectively for the first 3 years of life. Nasal samples were collected during acute infections in both cohorts and during asymptomatic periods in COAST and analyzed for RV-A, RV-B, and RV-C, and other common respiratory viruses. MEASUREMENTS AND MAIN RESULTS: The CDHR3 asthma risk allele (rs6967330-A) was associated with increased risk of respiratory tract illnesses (incidence risk ratio [IRR] = 1.14 [95% confidence interval, 1.05-1.23]; P = 0.003). In particular, this variant was associated with risk of respiratory episodes with detection of RV-C in COPSAC2010 (IRR = 1.89 [1.14-3.05]; P = 0.01) and in COAST (IRR = 1.37 [1.02-1.82]; P = 0.03) children, and in a combined meta-analysis (IRR = 1.51 [1.13-2.02]; P = 0.006). In contrast, the variant was not associated with illnesses related to other viruses (IRR = 1.07 [0.92-1.25]; P = 0.37). Consistent with these observations, the CDHR3 variant was associated with increased detection of RV-C, but not of other viruses during scheduled visits at specific ages. CONCLUSIONS: The CDHR3 asthma risk allele is associated specifically with RV-C illnesses in two birth cohorts. This clinical evidence supports earlier molecular evidence indicating that CDHR3 functions as an RV-C receptor, and raises the possibility of preventing RV-C infections by targeting CDHR3.

Prenatal vitamin D supplementation reduces risk of asthma/recurrent wheeze in early childhood: A combined analysis of two randomized controlled trials.

BACKGROUND: We recently published two independent randomized controlled trials of vitamin D supplementation during pregnancy, both indicating a >20% reduced risk of asthma/recurrent wheeze in the offspring by 3 years of age. However, neither reached statistical significance. OBJECTIVE: To perform a combined analysis of the two trials and investigate whether maternal 25-hydroxy-vitamin D (25(OH)D) level at trial entry modified the intervention effect. METHODS: VDAART (N = 806) and COPSAC2010. (N = 581) randomized pregnant women to daily high-dose vitamin D3 (4,000 IU/d and 2,400 IU/d, respectively) or placebo. All women also received a prenatal vitamin containing 400 IU/d vitamin D3. The primary outcome was asthma/recurrent wheeze from 0-3yrs. Secondary end-points were specific IgE, total IgE, eczema and lower respiratory tract infections (LRTI). We conducted random effects combined analyses of the treatment effect, individual patient data (IPD) meta-analyses, and analyses stratified by 25(OH)D level at study entry. RESULTS: The analysis showed a 25% reduced risk of asthma/recurrent wheeze at 0-3yrs: adjusted odds ratio (aOR) = 0.74 (95% CI, 0.57-0.96), p = 0.02. The effect was strongest among women with 25(OH)D level ≥30ng/ml at study entry: aOR = 0.54 (0.33-0.88), p = 0.01, whereas no significant effect was observed among women with 25(OH)D level <30ng/ml at study entry: aOR = 0.84 (0.62-1.15), p = 0.25. The IPD meta-analyses showed similar results. There was no effect on the secondary end-points. CONCLUSIONS: This combined analysis shows that vitamin D supplementation during pregnancy results in a significant reduced risk of asthma/recurrent wheeze in the offspring, especially among women with 25(OH)D level ≥ 30 ng/ml at randomization, where the risk was almost halved. Future studies should examine the possibility of raising 25(OH)D levels to at least 30 ng/ml early in pregnancy or using higher doses than used in our studies. TRIAL REGISTRATION: COPSAC2010: ClinicalTrials.gov NCT00856947; VDAART: ClinicalTrials.gov NCT00920621.

A rare IL33 loss-of-function mutation reduces blood eosinophil counts and protects from asthma.

IL-33 is a tissue-derived cytokine that induces and amplifies eosinophilic inflammation and has emerged as a promising new drug target for asthma and allergic disease. Common variants at IL33 and IL1RL1, encoding the IL-33 receptor ST2, associate with eosinophil counts and asthma. Through whole-genome sequencing and imputation into the Icelandic population, we found a rare variant in IL33 (NM_001199640:exon7:c.487-1G>C (rs146597587-C), allele frequency = 0.65%) that disrupts a canonical splice acceptor site before the last coding exon. It is also found at low frequency in European populations. rs146597587-C associates with lower eosinophil counts (ß = -0.21 SD, P = 2.5×10-16, N = 103,104), and reduced risk of asthma in Europeans (OR = 0.47; 95%CI: 0.32, 0.70, P = 1.8×10-4, N cases = 6,465, N controls = 302,977). Heterozygotes have about 40% lower total IL33 mRNA expression than non-carriers and allele-specific analysis based on RNA sequencing and phased genotypes shows that only 20% of the total expression is from the mutated chromosome. In half of those transcripts the mutation causes retention of the last intron, predicted to result in a premature stop codon that leads to truncation of 66 amino acids. The truncated IL-33 has normal intracellular localization but neither binds IL-33R/ST2 nor activates ST2-expressing cells. Together these data demonstrate that rs146597587-C is a loss of function mutation and support the hypothesis that IL-33 haploinsufficiency protects against asthma.

Shared genetic variants suggest common pathways in allergy and autoimmune diseases.

BACKGROUND: The relationship between allergy and autoimmune disorders is complex and poorly understood. OBJECTIVE: We sought to investigate commonalities in genetic loci and pathways between allergy and autoimmune diseases to elucidate shared disease mechanisms. METHODS: We meta-analyzed 2 genome-wide association studies on self-reported allergy and sensitization comprising a total of 62,330 subjects. These results were used to calculate enrichment for single nucleotide polymorphisms (SNPs) previously associated with autoimmune diseases. Furthermore, we probed for enrichment within genetic pathways and of transcription factor binding sites and characterized commonalities in variant burden on tissue-specific regulatory sites by calculating the enrichment of allergy SNPs falling in gene regulatory regions in various cells using Encode Roadmap DNase-hypersensitive site data. Finally, we compared the allergy data with those of all known diseases. RESULTS: Among 290 loci previously associated with 16 autoimmune diseases, we found a significant enrichment of loci also associated with allergy (P = 1.4e-17) encompassing 29 loci at a false discovery rate of less than 0.05. Such enrichment seemed to be a general characteristic for autoimmune diseases. Among the common loci, 48% had the same direction of effect for allergy and autoimmune diseases. Additionally, we observed an enrichment of allergy SNPs falling within immune pathways and regions of chromatin accessible in immune cells that was also represented in patients with autoimmune diseases but not those with other diseases. CONCLUSION: We identified shared susceptibility loci and commonalities in pathways between allergy and autoimmune diseases, suggesting shared disease mechanisms. Further studies of these shared genetic mechanisms might help in understanding the complex relationship between these diseases, including the parallel increase in disease prevalence.

Fish Oil-Derived Fatty Acids in Pregnancy and Wheeze and Asthma in Offspring.

BACKGROUND: Reduced intake of n-3 long-chain polyunsaturated fatty acids (LCPUFAs) may be a contributing factor to the increasing prevalence of wheezing disorders. We assessed the effect of supplementation with n-3 LCPUFAs in pregnant women on the risk of persistent wheeze and asthma in their offspring. METHODS: We randomly assigned 736 pregnant women at 24 weeks of gestation to receive 2.4 g of n-3 LCPUFA (fish oil) or placebo (olive oil) per day. Their children formed the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) cohort and were followed prospectively with extensive clinical phenotyping. Neither the investigators nor the participants were aware of group assignments during follow-up for the first 3 years of the children's lives, after which there was a 2-year follow-up period during which only the investigators were unaware of group assignments. The primary end point was persistent wheeze or asthma, and the secondary end points included lower respiratory tract infections, asthma exacerbations, eczema, and allergic sensitization. RESULTS: A total of 695 children were included in the trial, and 95.5% completed the 3-year, double-blind follow-up period. The risk of persistent wheeze or asthma in the treatment group was 16.9%, versus 23.7% in the control group (hazard ratio, 0.69; 95% confidence interval [CI], 0.49 to 0.97; P=0.035), corresponding to a relative reduction of 30.7%. Prespecified subgroup analyses suggested that the effect was strongest in the children of women whose blood levels of eicosapentaenoic acid and docosahexaenoic acid were in the lowest third of the trial population at randomization: 17.5% versus 34.1% (hazard ratio, 0.46; 95% CI, 0.25 to 0.83; P=0.011). Analyses of secondary end points showed that supplementation with n-3 LCPUFA was associated with a reduced risk of infections of the lower respiratory tract (31.7% vs. 39.1%; hazard ratio, 0.75; 95% CI, 0.58 to 0.98; P=0.033), but there was no statistically significant association between supplementation and asthma exacerbations, eczema, or allergic sensitization. CONCLUSIONS: Supplementation with n-3 LCPUFA in the third trimester of pregnancy reduced the absolute risk of persistent wheeze or asthma and infections of the lower respiratory tract in offspring by approximately 7 percentage points, or one third. (Funded by the Lundbeck Foundation and others; ClinicalTrials.gov number, NCT00798226 .).

Incidence and Determinants of Ventilation Tubes in Denmark.

BACKGROUND AND OBJECTIVES: Many children are treated for recurrent acute otitis media and middle ear effusion with ventilation tubes (VT). The objectives are to describe the incidence of VT in Denmark during 1997-2011 from national register data, furthermore, to analyze the determinants for VT in the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) birth cohort. METHODS: The incidence of VT in all children under 16 years from 1997-2011 were calculated in the Danish national registries. Determinants of VT were studied in the COPSAC2010 birth cohort of 700 children. RESULTS: Nationwide the prevalence of VT was 24% in children aged 0 to 3 three years, with a significant increase over the study period. For all children 0-15 years, the incidence of VT was 35/1,000. In the VT population, 57% was male and 43% females. In the COPSAC2010 birth cohort, the prevalence of VT during the first 3 years of life was 29%. Determinants of VT were: maternal history of middle ear disease; aHR 2.07, 95% CI [1.45-2.96] and siblings history of middle ear disease; aHR 3.02, [2.11-4.32]. Paternal history of middle ear disease, presence of older siblings in the home and diagnosis of persistent wheeze were significant in the univariate analysis but the association did not persist after adjustment. CONCLUSION: The incidence of VT is still increasing in the youngest age group in Denmark, demonstrating the highest incidence recorded in the world. Family history of middle ear disease and older siblings are the main determinants for VT.

Large-scale benchmarking reveals false discoveries and count transformation sensitivity in 16S rRNA gene amplicon data analysis methods used in microbiome studies.

BACKGROUND: There is an immense scientific interest in the human microbiome and its effects on human physiology, health, and disease. A common approach for examining bacterial communities is high-throughput sequencing of 16S rRNA gene hypervariable regions, aggregating sequence-similar amplicons into operational taxonomic units (OTUs). Strategies for detecting differential relative abundance of OTUs between sample conditions include classical statistical approaches as well as a plethora of newer methods, many borrowing from the related field of RNA-seq analysis. This effort is complicated by unique data characteristics, including sparsity, sequencing depth variation, and nonconformity of read counts to theoretical distributions, which is often exacerbated by exploratory and/or unbalanced study designs. Here, we assess the robustness of available methods for (1) inference in differential relative abundance analysis and (2) beta-diversity-based sample separation, using a rigorous benchmarking framework based on large clinical 16S microbiome datasets from different sources. RESULTS: Running more than 380,000 full differential relative abundance tests on real datasets with permuted case/control assignments and in silico-spiked OTUs, we identify large differences in method performance on a range of parameters, including false positive rates, sensitivity to sparsity and case/control balances, and spike-in retrieval rate. In large datasets, methods with the highest false positive rates also tend to have the best detection power. For beta-diversity-based sample separation, we show that library size normalization has very little effect and that the distance metric is the most important factor in terms of separation power. CONCLUSIONS: Our results, generalizable to datasets from different sequencing platforms, demonstrate how the choice of method considerably affects analysis outcome. Here, we give recommendations for tools that exhibit low false positive rates, have good retrieval power across effect sizes and case/control proportions, and have low sparsity bias. Result output from some commonly used methods should be interpreted with caution. We provide an easily extensible framework for benchmarking of new methods and future microbiome datasets.