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Supplemental Material Online Supporting Material SUPPLEMENTAL MATERIAL A taxonomic signature of obesity in a large study of American adults Brandilyn A. Peters,1 Jean A. Shapiro,2 Timothy R. Church,3 George Miller,4,5,6 Chau Trinh-Shevrin,1,6 Elizabeth Yuen,7 Charles Friedlander,7 Richard B. Hayes,1,6 Jiyoung Ahn1,6 1Department of Population Health, New York University School of Medicine, New York, NY, USA 2Division of Cancer Prevention and Control, Centers for Disease Control and Prevention, Atlanta, GA, USA 3Division of Environmental Health Sciences, School of Public Health, University of Minnesota, Minneapolis, MN, USA 4Departments of Surgery and 5Cell Biology, New York University School of Medicine, New York, NY, USA 6NYU Perlmutter Cancer Center, New York University School of Medicine, New York, NY, USA 7Kips Bay Endoscopy Center, New York, NY, USA Online Supporting Material Supplemental Figure 1. (a, b, c) Richness, Shannon diversity index, and Evenness rarefaction curves in healthy-weight, overweight, obese class I, and obese class II-III participants. Rarefaction curves were estimated by taking the mean of the α-diversity indices averaged for each participant over 100 iterations at each rarefaction sequencing depth. (d) Principal coordinate analysis of the weighted UniFrac distances. Shapes outlined in black represent centroids for healthy-weight, overweight, and obese participants. (e) Partial constrained analysis of principal coordinates (CAP) based on the weighted UniFrac distance. BMI category was the constraining variable, and sex, age, polyp status, and study were conditioning variables. Online Supporting Material Supplemental Figure 2. (a) Boxplots of richness (number of OTUs) in relation to BMI in subgroups by study, polyp status, and sex. P- values were obtained from linear regression models with richness at 1,490 sequence reads per sample as the outcome, and BMI category as the main predictor. Study-stratified models were adjusted for age, sex, and polyp status; polyp-stratified models were adjusted for age, sex, and study; sex-stratified models were adjusted for age, study, and polyp status. (b) Partial CAP of the weighted UniFrac distance within sub-groups, with BMI category as the constraining variable, and conditioning variables as listed for (a). Shapes outlined in black represent centroids for healthy-weight, overweight, and obese participants. * p < 0.05 Online Supporting Material Supplemental Figure 3. Mean contributions of OTUs associated with obesity to abundance of KEGG orthologs for butyrate synthesis (K00929: butyrate kinase, K01034: acetate CoA/acetoacetate CoA-transferase alpha subunit). Abundance contribution was averaged over all participants for each OTU. Only OTUs associated with obesity (LRT q<0.05 and pHolm<0.05) are plotted; green points represent OTUs depleted in the obese, and orange points represent OTUs enriched in the obese. Circles with black outlines indicate OTUs with non-zero contribution to the abundance of the KEGG ortholog. Another butyrate synthesis KEGG ortholog, K01035 was excluded from the plot since none of the obesity-associated OTUs contributed to its abundance. Supplemental Figure 4. Flow chart of the study design. Online Supporting Material Supplemental Table 1. Obesity and overweight in relation to α- and β-diversity. F-test F-test Overweight vs. healthy- Obese II-III vs. healthy- b b Obese vs. healthy-weight Obese I vs. healthy-weight (3-cat.) (4-cat.) weight weight c c d d p p β (se) p (pHolm) β (se) p (pHolm) β (se) p (pHolm) β (se) p (pHolm) Richnessa 0.002 0.005 -9.87 (4.68) 0.04 (0.08) 6.47 (4.12) 0.12 (0.12) -9.72 (5.40) 0.07 (0.21) -10.13 (6.63) 0.13 (0.24) Shannon indexa 0.03 0.07 -0.11 (0.07) 0.11 (0.22) 0.06 (0.06) 0.28 (0.28) -0.11 (0.08) 0.14 (0.42) -0.10 (0.10) 0.29 (0.56) Evennessa 0.14 0.26 -0.01 (0.01) 0.22 (0.44) 0.00 (0.01) 0.43 (0.44) -0.01 (0.01) 0.22 (0.66) -0.01 (0.01) 0.51 (0.86) 2 c 2 c 2 d 2 d p p R (%) p (pHolm) R (%) p (pHolm) R (%) p (pHolm) R (%) p (pHolm) e 0.037 0.639 0.071 0.194 Weighted UniFrac 0.14 0.28 0.37 0.10 0.32 0.22 (0.074) (0.639) (0.213) (0.388) aParameters are from linear regression models with specified α-diversity metric (averged over 100 iterations of rarefied OTU table at 1,490 sequence reads/sample) as outcome. All models were adjusted for age, sex, polyp status, and study. bTest of global BMI category variable; 3-category indicates healthy-weight, overweight, and obese groups; 4-category indicates healthy-weight, overweight, obese I, and obese II-III groups. cp-values were adjusted with the Holm method for two pairwise comparisons: obese vs. healthy-weight and overweight vs. healthy-weight. dp-values were adjusted by with the Holm method for three pairwise comparisons: obese I vs. healthy-weight, obese II-III vs. healthy-weight, and overweight vs. healthy-weight. eParameters are from permutational MANOVA of weighted UniFrac distance using 'adonis' function (Vegan package, R); adjustment factors of age, sex, polyp status, and study were included in the model first before BMI category. Supplemental Table 2. Obesity in relation to α- and β-diversity in sub-groups by study, polyp status, and sex. CDC study (n=423) NYU study (n=176) Polyps (n=288) No polyps (n=311) Men (n=321) Women (n=278) β (se) p β (se) p β (se) p β (se) p β (se) p β (se) p Richnessa,c -9.13 (5.31) 0.09 -16.86 (10.58) 0.11 -7.44 (6.58) 0.26 -12.23 (6.71) 0.07 -4.99 (6.95) 0.47 -14.41 (6.41) 0.03 Shannon a,c -0.12 (0.08) 0.11 -0.13 (0.15) 0.39 -0.08 (0.09) 0.4 -0.14 (0.10) 0.15 -0.07 (0.10) 0.51 -0.15 (0.09) 0.09 index Evennessa,c -0.01 (0.01) 0.16 -0.01 (0.01) 0.67 0.00 (0.01) 0.54 -0.01 (0.01) 0.26 0.00 (0.01) 0.59 -0.01 (0.01) 0.19 R2 (%) p R2 (%) p R2 (%) p R2 (%) p R2 (%) p R2 (%) p Weighted b,c 0.38 0.15 0.95 0.132 0.56 0.125 0.46 0.189 0.39 0.229 0.65 0.106 UniFrac aParameters are for obese vs. healthy-weight comparison in linear regression models with specified α-diversity metric (averaged over 100 iterations of rarefied OTU table at 1,490 sequence reads/sample) as outcome. bParameters are for obese vs. healthy-weight comparison in permutational MANOVA of UniFrac distances using 'adonis' function (Vegan package, R); adjustment factors of age, sex, polyp status, and study were included in the model first before BMI category. cStudy-stratified models were adjusted for age, sex, and polyp status; polyp-stratified models were adjusted for age, sex, and study; sex-stratified models were adjusted for age, study, and polyp status. Online Supporting Material Supplemental Table 3. Obesity in relation to α- and β-diversity after adjustment for dietary factors or exercise in the NYU study. No diet Total energy Fiber a,c Protein No exercise Exercise a a b a,c Fat adjustment a,c d d,e adjustment adjustment , adjustment adjustment adjustment adjustment β (se) p β (se) p β (se) p β (se) p β (se) p β (se) p β (se) p f -15.91 -14.61 Richness -15.18 (10.86) 0.16 0.14 -15.47 (10.79) 0.15 0.18 -15.72 (10.95) 0.15 -19.45 (10.75) 0.07 -24.26 (11.53) 0.04 (10.77) (10.83) Shannon -0.10 (0.15) 0.52 -0.11 (0.15) 0.48 -0.11 (0.15) 0.49 -0.09 (0.15) 0.57 -0.10 (0.16) 0.52 -0.16 (0.15) 0.3 -0.24 (0.16) 0.14 indexf Evennessf 0.00 (0.01) 0.81 0.00 (0.01) 0.78 0.00 (0.01) 0.78 0.00 (0.01) 0.89 0.00 (0.01) 0.84 -0.01 (0.01) 0.56 -0.02 (0.01) 0.29 R2 (%) p R2 (%) p R2 (%) p R2 (%) p R2 (%) p R2 (%) p R2 (%) p Weighted 0.86 0.155 0.88 0.164 0.86 0.152 0.83 0.173 0.83 0.184 1.02 0.101 1.10 0.097 UniFracg aModels were examined in n=171 NYU participants with diet data; all models were adjusted for age, sex, and polyp status; model parameters shown are for obese vs. healthy-weight comparison. bTotal energy intake (kcal) was added to the model. cFiber, fat, or protein was added to the model; these models also adjust for total energy intake. dModels were examined in n=175 NYU participants with exercise data; all models were adjusted for age, sex, and polyp status; model parameters shown are for obese vs. healthy-weight comparison. eExercise was added to the model. fThese α-diversity indices were the outcomes in linear regression models. gPermutational MANOVA of the UniFrac distances using 'adonis' function (Vegan package, R); all adjustment factors were included in the model first before BMI category. Online Supporting Material Supplemental Table 4. Differentially abundant taxa between obese and healthy‐weight participants, and overweight and healthy‐weight participants, as detected by the DESeq function in the DESeq2 package. Models were adjusted for age, sex, polyp status, and study.
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