traits and fasdng TMAO concentradons..6

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1 FIGURE S1: Variability of gut microbiota composidon in Metsim samples 2 FIGURE S2: AssociaDon of bacterial diversity and richness measures with traits.3 FIGURE S3: AssociaDons of OTUs with fasdng blood levels of metabolites. 4 FIGURE S4: Boxplots of glutamine and branched chain amino acid (BCAA) levels.5 FIGURE S5: CorrelaDon heatmap demonstradng the associadon between the metabolic traits and fasdng TMAO concentradons..6 FIGURE S6: Gut microbiota profile in obese and type 2 diabetes...7 FIGURE S7: Comparison between pret2d and NGS OTU networks

2 Figure S1: Variability of gut microbiota composi<on in Metsim samples. Columns represent the reladve abundance of microbial phyla on 538 Finnish males (A) and reladve abundance of microbial family on 13 replicated samples (B). The average reladve abundance for the top ten most prevalent bacterial family (C). A. C. Clostridiaceae S24-7 Porphyromonadaceae Rikenellaceae Prevotellaceae Veillonellaceae Clostridiales.Unkn Lachnospiraceae Ruminococcaceae Bacteroidaceae Relative abundance (%) 100%# 90%# 80%# Rela%ve'Abundance' 70%# 60%# 50%# 40%# 30%# 20%# 10%# 0%# 860# 860b# 1126# 1126b# 1251# 1251b# 1263# 1263b# 1288# 1288b# 1311# 1311b# 4321# 4321b# 4839# 4839b# 4921# 4921b# 5254# 5254b# 5259# 5259b# 5297# 5297b# 5569# 5569b# Methanobacteriaceae# Other# Bifidobacteriaceae# Coriobacteriaceae# Bacteroidales;Other# Bacteroidaceae# Porphyromonadaceae# Prevotellaceae# Rikenellaceae# S24H7# Barnesiellaceae# Odoribacteraceae# Paraprevotellaceae# YS2;Other# Lactobacillaceae# Streptococcaceae# #Turicibacteraceae# #Clostridiales;Other# Christensenellaceae# Clostridiaceae# Lachnospiraceae# Peptococcaceae# Ruminococcaceae# Veillonellaceae# Mogibacteriaceae# Tissierellaceae# Erysipelotrichaceae# Fusobacteriaceae# VicQvallaceae# RF32;Other# Alcaligenaceae# Desulfovibrionaceae# Helicobacteraceae# Enterobacteriaceae# Pasteurellaceae# Anaeroplasmataceae# RF39;Other# ML615JH28;Other# Verrucomicrobiaceae# 2

3 A. Figure S2: Associa<on of bacterial diversity and richness measures with traits. The heatmap shows the spearman correladon of each trait with alpha, richness (observed number of OTUs) and Shannon s index of diversity (A). The color key for correladon is shown. Significant differences in bacteria diversity and richness between groups with different HbA1C, glutamine and acetate levels (FDR<0.05)(B). Groups are divided as high or low based on mean metabolite concentradons. Color Key Value Shannon Hba1c (fdr=0.005) alpha richness shannon alpha (fdr=1.37e 04) richness Observed OTUs Observed OTUs shannon (fdr=0.037) nmr_la_fa nmr_faw6_fa nmr_pufa_fa nmr_dha_fa nmr_faw3_fa nmr_val nmr_unsat nmr_ace nmr_crea nmr_acace nmr_his nmr_gln hba1c nmr_dha nmr_ldl_c nmr_alb nmr_faw3 nmr_sm age nmr_sfa_fa nmr_ile nmr_glc nmr_tyr nmr_hdl_tg nmr_serum_c nmr_la nmr_faw6 nmr_vldl_c nmr_pufa nmr_apob nmr_cit nmr_bohbut nmr_hdl_c BMI HOMAIR Insulin nmr_leu nmr_totcho Fat nmr_apoa1 whr nmr_phe nmr_lac nmr_gp nmr_gly nmr_mufa_fa nmr_mufa nmr_glol nmr_pc nmr_totpg nmr_vldl_tg nmr_serum_tg nmr_sfa nmr_ldl_tg nmr_pyr nmr_ala nmr_totfa 3

4 Figure S3: Associa<ons of OTUs with fas<ng blood levels of metabolites. Significant associadons of circuladng serum metabolites with gut mirobiota OTUs (shared 50% of subjects) at FDR<0.05. AssociaDons were adjusted for age and treatment. Amino acids Isoleucine Isoleucine Leucine Leucine Leucine Phenylalanine Blautia_ Ruminococcaceae_ Ruminococcaceae_ Clostridiales Clostridiales_ Lachnospiraceae_ Lachnospiraceae_ Ruminococcaceae_ Ruminococcaceae_ Ruminococcus Ruminococcus torques_ Ruminococcus gnavus Glycolysis related metabolites Glucose Coriobacteriaceae Clostridiales_ Oscillospira_ Ruminococcus torques_ Lactate Lactate Clostridiales_ Lactate Ruminococcaceae_ Blautia Blautia_ Lachnospiraceae_ Ruminococcaceae_ Ruminococcus gnavus 0 Fatty Acids Actinobacteria Anaerostipes Bacteroidales Bacteroides Bifidobacterium longum_ Lachnospiraceae_ fatty acids Bacteroidales fatty acids Bifidobacterium longum_ fatty acids Lachnospiraceae_ MUFA Clostridiales_ MUFA MUFA 0 Inflammation & Traits HbA1C HbA1C HbA1C Clostridiales Clostridiales_ Coprococcus Lachnospira_ Oscillospira_ Ruminococcaceae_ Ruminococcaceae_ Ruminococcus Clostridiales_ Ruminococcaceae_ Fatty acids, relative to total fatty acids & Lipids (%) Actinobacteria (%) Anaerostipes (%) Bacteroides_ (%) Bifidobacterium (%) Bifidobacterium adolescentis (%) Bifidobacterium adolescentis_ (%) Bifidobacterium longum_ fatty acids (%) Ruminococcaceae_ fatty acids (%) fatty acids (%) fatty acids (%) Clostridiales_ fatty acids (%) Lachnospiraceae_ fatty acids (%) Lachnospiraceae_ fatty acids (%) Ruminococcus gnavus Clostridiales_ Clostridiales_ Coprococcus_ Lachnospiraceae_ Oscillospira_ Ruminococcaceae_ Ruminococcus torques_ Clostridiales Clostridiales_ Lachnospiraceae_ Lachnospiraceae_ Ruminococcaceae_ Blautia Lachnospiraceae_ Roseburia_ Ruminococcaceae_ Ruminococcus torques_ Saturated fatty acids (%) Dorea Saturated fatty acids (%) Lachnospiraceae Saturated fatty acids (%) Lachnospiraceae_ Saturated fatty acids (%) Ruminococcaceae_ Saturated fatty acids (%) Ruminococcaceae_ Unsaturation degree Ruminococcaceae_ VLDL-tg Lachnospiraceae_ Ketone bodies Other metabolites Carnitine Choline Lachnospira Lachnospira_ Lachnospira_ Ruminococcus gnavus Barnesiellaceae Barnesiellaceae_ Blautia Blautia_ Christensenellaceae Clostridiaceae Clostridiaceae_ Clostridiaceae_ Clostridiales_ Coprococcus_ ML615J_28_ Oscillospira_ Oscillospira_ Peptostreptococcaceae_ RF39_ Ruminococcaceae_ Ruminococcaceae_ Ruminococcus SMB53 SMB53_ SMB53_ Tenericutes Lachnospiraceae Lachnospiraceae_ Lachnospiraceae_ Lachnospiraceae_ Ruminococcaceae_ Lachnospiraceae_ Clostridiaceae_

5 Figure S4: Boxplots of glutamine and branched chain amino acid (BCAA) levels in subjects with low and high BMI (A) and subjects in different HOMA-IR levels (B). P values were obtained using Tukey s HSD test of significance. A. (mmol/l) BMI (p=0.009) Isoleucine (mmol/l) BMI (p=1.919e 07) Leucine (mmol/l) BMI (p= ) Valine (mmol/l) BMI (p= ) (mmol/l) HOMAIR Isoleucine (mmol/l) HOMAIR (p=4.034e 07) Leucine (mmol/l) HOMAIR (p=3.305e 06) Valine (mmol/l) HOMAIR (3.305e 06) 5

6 Figure S5: Correla<on heatmap demonstra<ng the associa<on between the metabolic traits and fas<ng TMAO concentra<ons. Coloring represents the Spearman correladon coefficient between TMAO and traits. FDRs are denoted: *, FDR < 0.01; * *, FDR < **FDR<= 0.001,*FDR<= 0.01 BMI Insulin HOMA IR HbA1C Apoa1 Triglyceride total cholesterol HDL cholesterol valine isoleucine leucine tyrosine glutamine histidine phenylalanine alanine glycine lactate unsataturated FA omega 3 FA MUFA Fat PUFA saturated FA total FA omega 6 FA creatinine albumine GlycA citrate glucose pyruvate glycerol acetate acatoacetate 3 hydroxybutyrate tmao * * ** *

7 Figure S6: Gut microbiota profile in obese and type 2 diabetes. Boxplot of β diversity distances (unweighted UniFrac) between microbial communides (A), RaDo of Firmicutes to Bacteoridetes individuals in different BMI classes (BMI<25, BMI and BMI>30 and between individuals based on glucose tolerance test (NGT, pret2d and newt2d) (B). Boxplot of HOMAIR and glycoprotein acetyls levels between individuals based on oral glucose tolerance test (OGTT) (C). * p< 0.01, **p< for Tukey HSD test. OGTT descripdons see Methods. A. BMI BMI Firmicutes/Bacteroides ratio Firmicutes/Bacteroides ratio (log) Shannon Shannon medium NGT pret2d T2D C. medium NGT pret2d T2D 0 2 IFG&IGT IsolIFG IsolIGT NEWT2DM NGT (mmol/l) IFG&IGT IsolIFG IsolIGT NEWT2DM NGT 7

8 Figure S7: Comparison between pret2d and NGS OTU networks A: OTU Co-occurrence networks for pret2d or NGS individuals. OTU (nodes) are colored by their corresponding WGCNA module colors. Edges are represented only for significant correladon between 2 OTU (P-value <0.01, abs (cor)>0.3). In both networks OTU are at the same locadons. Network layout was based on OTU co-occurrence in pret2d condidon. B: Module-trait associadon: each cell of the matrix contains the correladon between one OTU module and a metabolic trait, and the corresponding p-value. The table is color-coded by correladon according to the color legend (red for posidve correladons and green for negadve correladons). Only traits associated with at least one module were represented (P-value<0.01). C. Zsummary stadsdcs of module preservadon of pret2d OTU modules in NGS OTU modules (y-axis) vs. module size (x-axis). Green dashed line corresponds to the cutoff for very week preservadon and the blue one to no preservadon. D. Heatmap of the correladons between OTU abundance in tan module and glucose level in pret2d and NGS individuals. Circles sizes are propordonal to correladon values. Scamerplot is represented for OTU in pret2d and NGS condidons. 8

9 A. pret2d - OTU network NGS - OTU network C. D. 9