Basal diet determined long-term composition of the gut microbiome and mouse phenotype to a greater extent than fecal microbiome transfer from lean or obese human donors

Daphne M. Rodriguez1, Abby D. Benninghoff1,3*, Niklas D.J. Aardema2, Sumira Phatak1, and Korry J. Hintze2,3*

1. Department of Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322, USA 2. Department of Nutrition, Dietetics and Food Sciences, Utah State University, Logan, UT 84322, USA 3. USTAR Applied Nutrition Research, Utah State University, Logan, UT 84322, USA * Corresponding author: Abby D. Benninghoff, Utah State University, Department of Animal, Dairy and Veterinary Sciences, 4815 Old Main Hill, Logan, UT 84322-4815, USA, Phone: 435-797-8649, Email: [email protected], Korry J. Hintze, Utah State University, Department of Nutrition, Dietetics and Food Sciences, 8700 Old Main Hill, Logan, UT 84322-8700, USA, Phone: 435-797-2124, Email: [email protected]

SUPPLEMENTAL MATERIALS

Table S1 Parameters of donors Donor Age BMI Waist circumference (cm) Ethnicity Sex Lean L1 65 22.5 89.2 White Male L2 32 20.1 82.7 White Male L3 29 22.6 75.0 Asian Male Obese O4 28 34.8 113.7 White Male O5 28 30.6 101.5 White Male O6 65 31.0 109.5 White Male Abbreviations: BMI body mass index

Table S2 Composition of experimental diets

Nutrient AIN93G DIO TWD

Energy density (kcal/g) 3.8 4.6 4.4 Macronutrient Carbohydrates (g/kg diet) Corn starch 398 85 230 Maltodextrin 132 115 70 Sucrose 100 200 261 Cellulose 50 58 30 kcal (% of total) 60.1% 36.2% 54.5% Proteins (g/kg) Casein 200 245 190 L-cystine 3 3.5 2.8 kcal (% of total) 18.8% 19.0% 15.4% Fats (g/kg) Soybean oil 70 30 31.4 Anhydrous milk fat 36.3 Olive oil 28.0 Lard 195 28.0 Beef tallow 24.8 Corn oil 16.5 Cholesterol 0.4 kcal (% of total) 17.2% 44.8% 34.5% Micronutrients Minerals (mg/kg) Calcium 5000 5000 2011 Phosphorus 3000 3000 2757 Sodium 1019 1019 7078 Potassium 3600 3600 5333 Magnesium 507 507 589 Iron 35 35 31 Zinc 30 30 25 Copper 6 6 2.6 Selenium 0.15 0.15 0.2 Vitamins (unit/kg) Thiamin (mg) 5 5 3.5 Riboflavin (mg) 6 6 4.4 Niacin (mg) 30 30 50.6 Pyridoxine (mg) 6 6 3.9 Folate (mg) 2 2 1.3 Vitamin B12 (µg) 25 25 11 Vitamin A (IU) 4000 4000 4300 Vitamin D (IU) 1000 1000 391 Vitamin E (IU) 75 75 24.6 Vitamin K (µg) 750 750 189 Choline (mg) 1027 1027 648 Abbreviations: AIN American Institution of Nutrition, DIO diet-induced obesity, TWD total Western diet. Notes: Composition of the TWD was published previously [66] No data are available in NHANES for chloride, manganese, iodine, pantothenic acid, biotin, or ultra-trace minerals. Thus, levels of these components in the AIN93G diet were used in the formulation of all experimental diets.

Table S3 Primer sequences and barcodes for microbiome sequencing 1 Barcode Primer sequence (5’-3’) MIDD AACCAACC CCTACGGGRSGCAGCAG 1 AACCAAGG CCTACGGGRSGCAGCAG 2 AACCATCG CCTACGGGRSGCAGCAG 3 AACCATGC CCTACGGGRSGCAGCAG 4 AACCGCAT CCTACGGGRSGCAGCAG 5 AACCGCTA CCTACGGGRSGCAGCAG 6 AACCGGAA CCTACGGGRSGCAGCAG 7 AACCGGTT CCTACGGGRSGCAGCAG 8 AACCTACG CCTACGGGRSGCAGCAG 9 AACCTAGC CCTACGGGRSGCAGCAG 10 AACCTTCC CCTACGGGRSGCAGCAG 11 AACCTTGG CCTACGGGRSGCAGCAG 12 AACGAACG CCTACGGGRSGCAGCAG 13 AACGAAGC CCTACGGGRSGCAGCAG 14 AACGATCC CCTACGGGRSGCAGCAG 15 AACGATGG CCTACGGGRSGCAGCAG 16 AACGCCAT CCTACGGGRSGCAGCAG 17 AACGCCTA CCTACGGGRSGCAGCAG 18 AACGCGAA CCTACGGGRSGCAGCAG 19 AACGCGTT CCTACGGGRSGCAGCAG 20 AACGGCAA CCTACGGGRSGCAGCAG 21 AACGGCTT CCTACGGGRSGCAGCAG 22 AACGTACC CCTACGGGRSGCAGCAG 23 AACGTAGG CCTACGGGRSGCAGCAG 24 AACGTTCG CCTACGGGRSGCAGCAG 25 AACGTTGC CCTACGGGRSGCAGCAG 26 AAGCAACG CCTACGGGRSGCAGCAG 27 AAGCAAGC CCTACGGGRSGCAGCAG 28 AAGCATCC CCTACGGGRSGCAGCAG 29 AAGCATGG CCTACGGGRSGCAGCAG 30 AAGCCGAA CCTACGGGRSGCAGCAG 31 AAGCCGTT CCTACGGGRSGCAGCAG 32 AAGCGCAA CCTACGGGRSGCAGCAG 33 AAGCGCTT CCTACGGGRSGCAGCAG 34 AAGCGGAT CCTACGGGRSGCAGCAG 35 AAGCGGTA CCTACGGGRSGCAGCAG 36 AAGCTACC CCTACGGGRSGCAGCAG 37 AAGCTAGG CCTACGGGRSGCAGCAG 38 AAGCTTCG CCTACGGGRSGCAGCAG 39 AAGCTTGC CCTACGGGRSGCAGCAG 40 AAGGAACC CCTACGGGRSGCAGCAG 41 AAGGAAGG CCTACGGGRSGCAGCAG 42 AAGGATCG CCTACGGGRSGCAGCAG 43 AAGGATGC CCTACGGGRSGCAGCAG 44 AAGGCCAA CCTACGGGRSGCAGCAG 45 AAGGCCTT CCTACGGGRSGCAGCAG 46 AAGGCGAT CCTACGGGRSGCAGCAG 47 AAGGCGTA CCTACGGGRSGCAGCAG 48 AAGGTACG CCTACGGGRSGCAGCAG 49 AAGGTAGC CCTACGGGRSGCAGCAG 50 AAGGTTCC CCTACGGGRSGCAGCAG 51 AAGGTTGG CCTACGGGRSGCAGCAG 52 AATACCGC CCTACGGGRSGCAGCAG 53 Table S3 Primer sequences and barcodes for microbiome sequencing 1 Barcode Primer sequence (5’-3’) MIDD AATACGCC CCTACGGGRSGCAGCAG 54 AATAGCGG CCTACGGGRSGCAGCAG 55 AATAGGCG CCTACGGGRSGCAGCAG 56 AATTCCGG CCTACGGGRSGCAGCAG 57 AATTCGCG CCTACGGGRSGCAGCAG 58 AATTCGGC CCTACGGGRSGCAGCAG 59 AATTGCCG CCTACGGGRSGCAGCAG 60 1 Ion Torrent Primer A-Key CCATCTCATCCCTGCGTGTCTCCGAC; Ion Torrent Primer P-1 Key CCTCTCTATGGGCAGTCGGTGAT; key sequence TCAG; linker GAT; reverse primer sequence was ATTACCG.

A B 700 3500

3000 600 ** ns ns ns ** 2500 500 ns 2000 400 Total food intake (g) intake food Total 1500 Total energy intake (kcal) intake energy Total 0 0 Ln Ob Ln Ob Ln Ob Ln Ob Ln Ob Ln Ob AIN DIO TWD AIN DIO TWD * ns **** * * **** Mixed model main effects p value Mixed model main effects p value Diet 0.0090 Diet <0.0001 Body type 0.0490 Body type 0.0490 Diet x body type 0.0080 Diet x body type 0.0080 Donor ID[body type] 0.7502 Donor ID[body type] 0.7502

Fig. S1 Food and energy intake. Data are presented in Tukey box-plots (box, 25th to 75th percentiles; whiskers, 1.5 IQR; +, mean) (n = 6 to 9) for total food intake (A) and total energy intake (B). The table beneath each panel shows p values for main effects of each experimental factor as determined by the mixed model analysis. Below the plot, brackets indicate results of Tukey post-hoc tests for overall effects of each diet. Within each diet group, symbols above the box-and-whisker bars indicate results of post-hoc tests comparing mice that received bacteria from lean (Ln) or obese (Ob) human donors. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns=not significant.

A 2.5 B 1.2

2.0 ns ns ns ns 0.8 ns 1.5 *

1.0 0.4 Liver Weight (g)

0.5 Cecum weight (g)

0.0 0.0 Ln Ob Ln Ob Ln Ob Ln Ob Ln Ob Ln Ob AIN DIO TWD AIN DIO TWD ns ns ns ns ns ns

Mixed model main effects p value Mixed model main effects p value Diet 0.1398 Diet 0.1000 Body type 0.0485 Body type 0.0439 Diet x body type 0.9730 Diet x body type 0.2228 Donor ID[body type] 0.5954 Donor ID[body type] 0.1740

Fig. S2 Liver and cecum weights. Data for liver weight (A) and cecum weight (B) are shown in Tukey box-plots (box, 25th to 75th percentiles; whiskers, 1.5 IQR; +, mean) (n = 6 to 9). The tables below each panel show p values for main effects of each experimental factor as determined by the mixed model analysis. Below each plot, brackets indicate results of Tukey post-hoc tests for overall effects of each diet. Within each diet group, symbols above the box- and-whisker bars indicate results of post-hoc tests comparing mice that received bacteria from lean (Ln) or obese (Ob) human donors. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001, ns=not significant.

Fig. S3 Heatmap depicting bidirectional, unsupervised hierarchical cluster analysis of initial mouse fecal microbiomes. Values shown are the normalized reads for all mapped OTUs in the initial fecal mouse microbiomes, prior to antibiotic or diet treatments. Clustering was performed using Euclidian distance with average linkage. Heatmap color scale indicates relative abundance calculated as the log10 reads per million for better visualization of lower abundance OTUs.

AIN diet

1.0

0.8

0.6

0.4

0.2 Relative proportion of mapped reads

0.0 L1.A1 L1.A2 L1.A3 L1.A4 L1.A5 L1.A6 L2.A1 L2.A2 L2.A3 L2.A4 L2.A5 L2.A6 L2.A7 L2.A8 L2.A9 L3.A1 L3.A2 L3.A3 L3.A4 L3.A5 L3.A6 L3.A7 O4.A1 O4.A2 O4.A3 O4.A4 O4.A5 O4.A6 O4.A7 O5.A1 O5.A2 O5.A3 O5.A4 O5.A5 O5.A6 O6.A1 O6.A2 O6.A3 O6.A4 O6.A5 O6.A6 O6.A7

Bifidobacteriaceae Actinobacteria DIO diet Coriobacteriaceae 1.0 Prevotellaceae Paraprevotellaceae Odoribacteraceae Rikenellaceae Bacteroidetes 0.8 Barnesiellaceae Porphyromonadaceae Bacteroidaceae Enterococcaceae 0.6 Streptococcaceae Turicibacteraceae Clostridiales unclassified Clostridiaceae 0.4 Christensenellaceae Firmicutes Veillonellaceae Eubacteriaceae Lachnospiraceae 0.2 Peptostreptococcaceae Relative proportion of mapped reads Ruminococcaceae Erysipelotrichaceae

0.0 Enterobacteriaceae Alcaligenaceae Proteobacteria Desulfovibrionaceae L1.D1 L1.D2 L1.D3 L1.D4 L1.D5 L1.D6 L1.D7 L1.D8 L2.D1 L2.D2 L2.D3 L2.D4 L2.D5 L2.D6 L3.D1 L3.D2 L3.D4 L3.D5 L3.D6 L3.D7 O4.D1 O4.D2 O4.D3 O4.D4 O4.D5 O4.D6 O4.D7 O5.D1 O5.D2 O5.D3 O5.D4 O5.D5 O5.D6 O5.D7 O5.D8 O6.D1 O6.D2 O6.D3 O6.D4 O6.D5 O6.D6 O6.D7 Verrucomicrobiaceae Verrucomicrobia Anaeroplasmataceae Tenericutes TWD diet

1.0

0.8

0.6

0.4

0.2 Relative proportion of mapped reads

0.0 L1.T1 L1.T2 L1.T3 L1.T4 L1.T5 L1.T6 L1.T7 L2.T1 L2.T2 L2.T3 L2.T4 L2.T5 L2.T6 L3.T1 L3.T2 L3.T3 L3.T4 L3.T5 L3.T6 L3.T7 L3.T8 O4.T2 O4.T3 O4.T4 O4.T6 O4.T7 O5.T1 O5.T2 O5.T3 O5.T4 O5.T6 O5.T7 O5.T8 O6.T1 O6.T2 O6.T3 O6.T4 O6.T5 O6.T6 O6.T7 Fig. S4 Classification of human or mouse fecal bacteria by family for individual mice post fecal microbiome transfer. Data shown are the relative abundance of bacteria to annotated taxa (phylum and family) for individual mice for each experimental diet group. Mouse identification codes that begin with L indicate recipients of FMT from lean human donors, and codes that begin with O indicate recipients of FMT from obese human donors. Post FMT: Body type Post FMT: Diet

Actinobacteria (p) Collinsella aerofaciens Bacteroidetes (p) Bacteroides ovatus Barnesiellaceae (f) Butyricimonas (g) Paraprevotella (g) Firmicutes (p) Lactococcus (g) Christensenellaceae (f) Clostridiales (o) Caloramator (g) Pseudoramibacter_Eubacterium (g) Lachnospiraceae (f) Blautia producta Coprococcus (g) Dorea (g) Ruminococcus (g) Mogibacteriaceae (f) Holdemania (g) Proteobacteria (p) Bilophila (g) Enterobacteriaceae (f) Verrucomicrobia (p) Akkermansia muciniphila

0 -6 -5 -4 -3 -2 -1 0 0 -6 -5 -4 -3 -2 -1 0 Relative abundance Relative abundance Lean Obese AIN DIO TWD

Fig. S5 Relative abundance of selected bacteria taxa by main factors, body type and experimental diet, post fecal microbiome transfer. Data shown are individual relative abundance values for each mouse, with the black bar and whiskers representing the mean ± SEM on a log10 scale to facilitate visualization of less abundant species. Bold titles indicate the major represented phyla, with members of interest listed below.

0 -1 -2 Obese -3 -4 Lean Relative abundance -5 Post FMT: TWD diet -6 0 0 -1 -2 Obese -3 -4 Lean Relative abundance -5 Post FMT: DIO diet -6 0 0 -1 -2 Obese -3 -4 Lean Relative abundance -5 Post FMT: AIN diet -6 0 Dorea (g) Dorea Bilophila (g) Bilophila Firmicutes Firmicutes (p) Holdemania (g) Holdemania Clostridiales (o) Clostridiales Caloramator (g) Caloramator Lactococcus (g) Lactococcus Blautia producta Blautia Coprococcus (g) Coprococcus Butyricimonas (g) Butyricimonas Paraprevotella (g) Paraprevotella Ruminococcus (g) Ruminococcus Bacteroidetes (p) Bacteroidetes Barnesiellaceae (f) Barnesiellaceae Actinobacteria (p) Actinobacteria Bacteroides ovatus Bacteroides Proteobacteria (p) Proteobacteria Lachnospiraceae (f) Lachnospiraceae Mogibacteriaceae (f) Mogibacteriaceae Verrucomicrobia (p) Enterobacteriaceae (f) Enterobacteriaceae Collinsella aerofaciens Collinsella Christensenellaceae (f) Christensenellaceae Akkermansia muciniphila Akkermansia

Pseudoramibacter_Eubacterium (g) Pseudoramibacter_Eubacterium Fig. S6 Relative abundance of selected bacteria taxa in lean or obese recipient mice stratified by experimental diet post fecal microbiome transfer. Data shown are individual relative abundance values for each mouse, with the black bar and whiskers representing the mean ± SEM on a log10 scale to facilitate visualization of less abundant species. Bold titles indicate the major represented phyla, with members of interest listed below.

Fig. S7 Discriminating taxa for recipient mouse microbiomes post fecal transfer. LEfSe analyses were performed using relative abundance data averaged by human donor at the highest classification level taxonomy available. Data shown are the log10 linear discriminant analysis (LDA) scores following LEfSe analyses and the hierarch of discriminating taxa visualized as cladograms for the following class comparisons: (A) all lean donor recipient mice compared to all obese donor recipient mice, (B) all mice fed AIN diet compared to those fed DIO diet, and (C) all mice fed DIO diet compared to those fed TWD. (No discriminating features were identified for all mice fed AIN compared to those fed TWD.) The structure of these comparisons was designed to mimic tests for main effects of body type and diet (including diet pairwise tests), as was done for other study parameters using the linear mixed model. Relative abundance data of selected taxa of interest are shown in Fig. S4. AIN diet

1.0

0.8

0.6

0.4

0.2 Relative proportion of mapped reads

0.0 L1.A1 L1.A2 L1.A3 L1.A4 L1.A5 L1.A6 L1.A7 L2.A1 L2.A2 L2.A3 L2.A4 L2.A5 L2.A6 L2.A7 L3.A1 L3.A2 L3.A3 L3.A4 L3.A5 L3.A6 L3.A7 O4.A2 O4.A3 O4.A4 O4.A5 O4.A6 O4.A7 O5.A1 O5.A2 O5.A3 O5.A4 O5.A5 O5.A6 O6.A1 O6.A2 O6.A3 O6.A4 O6.A5 O6.A6 O6.A7 Bifidobacteriaceae Actinobacteria DIO diet Coriobacteriaceae Prevotellaceae 1.0 Paraprevotellaceae Odoribacteraceae Rikenellaceae Bacteroidetes Barnesiellaceae 0.8 Porphyromonadaceae Bacteroidaceae Enterococcaceae Streptococcaceae 0.6 Turicibacteraceae Clostridiales unclassified Clostridiaceae Christensenellaceae Firmicutes 0.4 Veillonellaceae Eubacteriaceae Lachnospiraceae Peptostreptococcaceae 0.2 Ruminococcaceae

Relative proportion of mapped reads Erysipelotrichaceae Enterobacteriaceae Alcaligenaceae Proteobacteria 0.0 Desulfovibrionaceae Verrucomicrobiaceae Verrucomicrobia L1.D1 L1.D2 L1.D3 L1.D4 L1.D5 L1.D6 L1.D7 L1.D8 L2.D1 L2.D2 L2.D3 L2.D4 L2.D5 L2.D6 L3.D1 L3.D2 L3.D4 L3.D5 L3.D6 L3.D7 O4.D1 O4.D2 O4.D3 O4.D4 O4.D5 O4.D6 O4.D7 O5.D1 O5.D2 O5.D3 O5.D4 O5.D5 O5.D6 O5.D7 O6.D1 O6.D2 O6.D3 O6.D4 O6.D5 O6.D6 O6.D7 Anaeroplasmataceae Tenericutes TWD diet

1.0

0.8

0.6

0.4

0.2 Relative proportion of mapped reads

0.0 L1.T1 L1.T2 L1.T3 L1.T4 L1.T5 L1.T6 L2.T1 L2.T2 L2.T3 L2.T4 L2.T5 L2.T6 L3.T1 L3.T2 L3.T3 L3.T4 L3.T5 L3.T6 L3.T7 L3.T8 O4.T2 O4.T3 O4.T4 O4.T6 O4.T7 O5.T1 O5.T2 O5.T3 O5.T4 O5.T6 O5.T7 O5.T8 O6.T1 O6.T2 O6.T3 O6.T4 O6.T5 O6.T6 O6.T7 Fig. S8 Classification of human or mouse fecal bacteria by family for individual mice at necropsy. Data shown are the relative abundance of bacteria to annotated taxa (phylum and family) for individual mice for each experimental diet group. Mouse identification codes that begin with L indicate recipients of FMT from lean human donors, and codes that begin with O indicate recipients of FMT from obese human donors.

Terminal: Body type Terminal: Diet

Actinobacteria (p) Eggerthella lenta Collinsella aerofaciens Adlercreutzia (g) Bacteroidetes (p) Barnesiellaceae (f) Butyricimonas (g) Paraprevotella (g) Firmicutes (p) Enterococus (g) Lactococcus (g) Turicibacter (g) Clostridiales (o) Christensenellaceae (f) Clostridiaceae (f) Caloramator (g) Pseudoramibacter_Eubacterium (g) Lachnospiraceae (f) Ruminococcus (g) Coprococcus (g) Dorea (g) Erysipelotrichales (o) Proteobacteria (p) Bilophila (g) Verrucomicrobia (p) Akkermansia muciniphila

0 -6 -5 -4 -3 -2 -1 0 0 -6 -5 -4 -3 -2 -1 0 Relative abundance Relative abundance Lean Obese AIN DIO TWD

Fig. S9 Relative abundance of selected bacteria taxa by main factors, body type and experimental diet, at necropsy. Data shown are individual relative abundance values for each mouse, with the black bar and whiskers representing the mean ± SEM on a log10 scale to facilitate visualization of less abundant species. Bold titles indicate the major represented phyla, with members of interest listed below.

0 -1 -2 Obese -3 -4 Lean Relative abundance -5 Terminal: TWD diet TWD Terminal: -6 0 0 -1 -2 Obese -3 -4 Lean Relative abundance -5 Terminal: DIO diet DIO Terminal: -6 0 0 -1 -2 Obese -3 -4 Lean Relative abundance -5 Terminal: AIN diet AIN Terminal: -6 0 Dorea (g) Dorea Bilophila (g) Bilophila Firmicutes Firmicutes (p) Turicibacter (g) Clostridiales (o) Clostridiales Caloramator (g) Caloramator Enterococus (g) Enterococus (g) Lactococcus Adlercreutzia (g) Adlercreutzia (g) Coprococcus Eggerthella lenta Eggerthella Clostridiaceae (f) Clostridiaceae Butyricimonas (g) Butyricimonas Paraprevotella (g) Paraprevotella Ruminococcus (g) Ruminococcus Bacteroidetes (p) Bacteroidetes Barnesiellaceae (f) Barnesiellaceae Actinobacteria (p) Actinobacteria Proteobacteria (p) Proteobacteria Lachnospiraceae (f) Lachnospiraceae Erysipelotrichales (o) Erysipelotrichales Verrucomicrobia (p) Collinsella aerofaciens Collinsella Christensenellaceae (f) Christensenellaceae Akkermansia muciniphila Akkermansia

Pseudoramibacter_Eubacterium (g) Pseudoramibacter_Eubacterium Fig. S10 Relative abundance of selected bacteria taxa in lean or obese recipient mice stratified by experimental diet at necropsy. Data shown are individual relative abundance values for each mouse, with the black bar and whiskers representing the mean ± SEM on a log10 scale to facilitate visualization of less abundant species. Bold titles indicate the major represented phyla, with members of interest listed below. A Terminal: Body type Clostridia Lean Prevotella Obese C Terminal: AIN vs TWD Prevotellaceae Eubacteriaceae Turicibacteraceae Pseudoramibacter_Eubacterium Turicibacterales Dorea Turicibacter Ruminococcus gnavus Clostridiaceae Bacteroides caccae Pseudoramibacter_Eubacterium Coriobacteriales 02d06 Coriobacteriia Caloramator 02d06 OPB54 Ruminococcus EtOH8 Coriobacteriaceae Bacteria Anaerostipes S24_7 Anaerotruncus Butyricimonas Mogibacteriaceae Adlercreutzia Odoribacteraceae Holdemania Bacilli Christensenellaceae Streptomycetaceae Bacilli Firmicutes Bacillales Bacteroides ovatus Unassigned Enterococcus Collinsella Allobaculum Barnesiellaceae Erysipelotrichi Collinsella aerofaciens Erysipelotrichales Verrucomicrobiae Verrucomicrobiales -10 -8 -6 -4 -2 0 2 4 6 8 10 Akkermansia muciniphila LDA score (log ) Akkermansia 10 AIN Verrucomicrobiaceae Verrucomicrobia TWD Moryella -10 -8 -6 -4 -2 0 2 4 6 8 10 Terminal: DIO vs TWD LDA score (log10) D

Turicibacteraceae Terminal: AIN vs DIO Turicibacterales B Turicibacter Lactococcus Clostridiaceae Streptococcaceae Dorea Clostridiales Enterococcaceae Clostridiaceae Enterococcus EtOH8 SMB53 Tenericutes Clostridiales Oscillospira Streptococcaceae Mollicutes Lactococcus Streptomycetaceae Coriobacteriaceae -10 -8 -6 -4 -2 0 2 4 6 8 10 Adlercreutzia LDA score (log ) S24_7 10 Allobaculum DIO TWD -10 -8 -6 -4 -2 0 2 4 6 8 10 LDA score (log ) 10 AIN A Terminal: Body type DIO Clostridia Lean Prevotella Obese C Terminal: AIN vs TWD Prevotellaceae Eubacteriaceae Turicibacteraceae Pseudoramibacter_Eubacterium Turicibacterales Dorea Turicibacter Ruminococcus gnavus Clostridiaceae Bacteroides caccae Pseudoramibacter_Eubacterium Coriobacteriales 02d06 Coriobacteriia Caloramator 02d06 OPB54 Ruminococcus EtOH8 Coriobacteriaceae Bacteria Anaerostipes S24_7 Anaerotruncus Butyricimonas Mogibacteriaceae Adlercreutzia Odoribacteraceae Holdemania Bacilli Christensenellaceae Streptomycetaceae Bacilli Firmicutes Bacillales Bacteroides ovatus Unassigned Enterococcus Collinsella Allobaculum Barnesiellaceae Erysipelotrichi Collinsella aerofaciens Erysipelotrichales Verrucomicrobiae Verrucomicrobiales -10 -8 -6 -4 -2 0 2 4 6 8 10 Akkermansia muciniphila LDA score (log ) Akkermansia 10 AIN Verrucomicrobiaceae Verrucomicrobia TWD Moryella -10 -8 -6 -4 -2 0 2 4 6 8 10 Terminal: DIO vs TWD LDA score (log10) D

Turicibacteraceae Terminal: AIN vs DIO Turicibacterales B Turicibacter Lactococcus Clostridiaceae Streptococcaceae Dorea Clostridiales Enterococcaceae Clostridiaceae Enterococcus EtOH8 SMB53 Tenericutes Clostridiales Oscillospira Streptococcaceae Mollicutes Lactococcus Streptomycetaceae Coriobacteriaceae -10 -8 -6 -4 -2 0 2 4 6 8 10 Adlercreutzia LDA score (log ) S24_7 10 Allobaculum DIO TWD -10 -8 -6 -4 -2 0 2 4 6 8 10 LDA score (log ) 10 AIN DIO

Fig. S11 Discriminating taxa for recipient mouse microbiomes at necropsy. LEfSe analyses were performed using relative abundance data averaged by human donor at the highest classification level taxonomy available. Data shown are the log10 linear discriminant analysis (LDA) scores following LEfSe analyses and the hierarch of discriminating taxa visualized as cladograms for the following class comparisons: (A) all lean donor recipient mice compared to all obese donor recipient mice, (B) all mice fed AIN diet compared to those fed DIO diet, and (C) all mice fed DIO diet compared to those fed TWD. (No discriminating features were identified for all mice fed AIN compared to those fed TWD.) The structure of these comparisons was designed to mimic tests for main effects of body type and diet (including diet pairwise tests), as was done for other study parameters using the linear mixed model. Relative abundance data of selected taxa of interest are shown in Fig. S6.

A B Source Original microbiomes Bacteroidales S24-7 Mouse Pre-AB 0.50 Mouse pre-AB Lactobacillaceae Lean donor Lean donor Streptococcaceae Veillonellaceae Obese donor Obese donor Bifidobacteriaceae Enterobacteriaceae 0.25 Alcaligenaceae Barnesiellaceae Verrucomicrobiaceae Turicibacteraceae 0.00 Anaeroplasmataceae Planococcaceae Clostridiaceae PC2 (23.3%) Odoribacteraceae Paraprevotellaceae -0.25 Coriobacteriaceae Porphyromonadaceae Fraction of total reads Erysipelotrichaceae Rikenellaceae Prevotellaceae -0.75 -0.50 -0.25 0.00 0.25 0.50 Bacteroidaceae PC1 (49.5%) Lachnospiraceae Clostridiales unknown Ruminococcaceae Mouse L3 O5 O4 O6 L1 L2

Fig. S12 Hierarchical clustering and principal components analysis of original mouse and human donor microbiomes. (A) Unsupervised, bidirectional hierarchical cluster analysis of taxonomy relative abundance data for bacteria families comprising a least 1% of the fecal microbiome from all mice prior to antibiotic treatment (pre-AB) and from each of the six human donors (lean: L1, L2, L3; obese, O4, O5, O6). Clustering was performed using Euclidian distance with average linkage. Heatmap color scale indicates relative abundance as fraction of total reads. (B) PCA of family-level taxonomy data plotted according to the first two principal components. Variation attributed to each component is shown. Dashed lines represent the 95% confidence ellipse.

A Post FMT - unweighted unifrac B Post FMT - weighted unifrac 0.4

0.2

0.0 0.0 Lean donors L1 PC2 (9.2%) PC2 (9.0%) -0.2 L2 L3 -0.5 p=0.001 p=0.001 -0.4 -0.2 0.0 0.2 0.4 -0.4 -0.2 0.0 0.2 0.4 0.6 Obese donors PC1 (12.3%) PC1 (32.2%) C D O4 Terminal - unweighted unifrac Terminal - weighted unifrac O5 0.2 0.4 O6

0.1 0.2

0.0 0.0

-0.1 -0.2 PC2 (6.6%) PC2 (21.0%) -0.2 -0.4

p=0.001 p=0.013 -0.3 -0.6 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 -0.4 -0.2 0.0 0.2 0.4 PC1 (7.4%) PC1 (24.8%)

E Post FMT - unweighted unifrac F Post FMT - weighted unifrac 0.4 0.4

0.2 0.2

0.0 0.0 -0.2 PC2 (9.2%) -0.2 PC2 (19.0%) -0.4

p=0.019 p=0.001 Experimental diets -0.4 -0.6 -0.2 0.0 0.2 0.4 -0.4 -0.2 0.0 0.2 0.4 0.6 AIN PC1 (12.8%) PC1 (32.2%) DIO TWD G Terminal - unweighted unifrac H Terminal - weighted unifrac 0.2 0.4

0.1 0.2

0.0 0.0

-0.1 -0.2 PC2 (6.7%) PC2 (21.1%) -0.2 -0.4

p=0.001 p=0.001 -0.3 -0.6 -0.3 -0.2 -0.1 0.0 0.1 0.2 0.3 -0.4 -0.2 0.0 0.2 0.4 PC1 (7.4%) PC1 (24.8%) Fig. S13 Beta diversity principal coordinates plots for weighted and unweighted unifrac distances for post FMT and terminal recipient mouse microbiomes. Data shown are the first two principal components of unweighted or weighted unifrac distances for post FMT and terminal fecal microbiomes categorized by human donor body type (A-D) or experimental diet (E-). Variation attributed to each coordinate is shown. Permanova p values are shown for each plot.