SUPPLEMENTARY INFORMATION
B
A
60
relative intensity of observed cholic acid
300
PC2 (R2 X=8.2%)
40
20
0
-20
-40
Sham
RYGB
p=0.012
p=0.087
p=0.4
200
100
-60
0
-100
-50
0
50
2
PC1 (R X=24.9%)
100
1 week pre-op
Week 6
Week 8
Sham
RYGB
400
p=0.001
p=0.026
p=0.7
200
0
1 week pre-op
Week 6
Week 8
relative intensity of observed taurine-conjugated bile acids
D
C
relative intensity of observed unconjugated bile acids
0
Sham
RYGB
p=0.073
p=0.049
Week 6
Week 8
2
1
p=0.3
0
1 week pre-op
S1. A PLS-DA scores plot (A, Q2Y=0.83) derived from UPLC-MS(-)data of faecal
samples shows a separation between 6 sham (blue) and 6 RYGB (red)-operated rats
at week 8. Plot (B) indicates cholic acid levels in both sham- and RYGB-operated rats
at 1 week pre-operation, 6 and 8 weeks post operation, which is the main
contributor to the group separation post surgery. Relative concentrations of
observed unconjugated (C) and taurine-conjugated (D) bile acids show a significant
difference between sham and RYGB-operated rats at weeks 6 and 8. Error bar
indicates standard error of the mean and the significance value, p, was calculated by
Student’s t-Test.
1
S2. Phylogenetic tree (neighbor-joining from a jukes-cantor matrix) of the main
taxonomic groups which varied between the two test groups, only the higher
taxonomic groups from which they were clustered is shown. The barchart to the
right of the tree shows the reads associated with a representative from its taxonomic
group. * Enterobacter hormaechei.
2
Crn HP
HP
A
Tau
Cre/Crn
Cit GT Suc
Ace
PAG
FA
IS
AP
Lac
FM
B
Ala Lac
BT
AP BT
PAG TMAO
IS
BT
DMA
PG
MA
HA
HA
PG AV
PS
PG
MNA
9.0
MNA
8.5
8.0
AV Ala Lac
MNA
7.5
7.0
4.5
4.0
3.5
3.0
2.0
1.5
1.0
ppm
S3. Typical 600 MHz 1H NMR spectra of urine obtained from a sham control rat (A)
and a Roux-en-Y Gastric Bypass (RYGB)-operated rat (B) 8-week post operation. The
spectra in the aromatic region (δ1H 6.4-9.3) and the region δ1H 0.6-2.28 were
magnified twice compared to the region δ1H 2.28-4.7. Keys: Ace: acetate; Ala:
alanine; AP: 2-oxoadipate; AV: 5-aminovalerate; BT: butyrate; Cit: citrate; Cre:
creatine; Crn: creatinine; DMA: dimethylamine; FA: formate; FM: fumarate; GT: 2oxoglutarate; HA: p-hydroxyphenylacetate; HP: hippurate; IS: indoxyl sulfate; Lac:
lactate; MA: methylamine; MNA: 1-methylnicotinamide; PAG: phenylacetylglycine;
PG: p-cresyl glucuronide; PS: p-cresyl sulfate; Suc: succinate; Tau: taurine; TMAO:
trimethylamine N-oxide.
3
A
OS/Amino acids
Ace
Ura
Ura
BT
BT/PP
AV
AV
Asp
FA
B
Suc
Gly
MT
Ura
Phe Tyr
FA
Ura
8.0
7.5
6.5
4.5
Val/Ileu/Leu/PP
GABA
Glu
BT
Lac
7.0
GABA
PT
Ala Lac
DE
TMA MA
4.0
3.5
3.0
2.5
ET
2.0
1.5
1.0
ppm
4
S4. Typical 600 MHz 1H NMR spectra of faecal extracts obtained from a sham control
rat (A) and a Roux-en-Y Gastric Bypass (RYGB)-operated rat (B) 8-week post
operation. The spectra in the aromatic region (δ1H 5.75-8.5) were magnified 4 times
compared to the region δ1H 0.5-4.7. Keys: Ace: acetate; Ala: alanine; AP: 2oxoadipate; Asp: aspartate; AV: 5-aminovalerate; BT: butyrate; DE: diaminoethane;
ET: ethanol; FA: formate; FM: fumarate; GABA: -animo-N-butyrate; Glu: glutamate;
Gly: glycine; Ileu: isoleucine; Lac: lactate; Leu: leucine; MA: methylamine; MT:
methanol; OS: oligosaccharides; Phe: phenylalanine; PP: propionate; PT: putrescine;
Suc: succinate; TMA: trimethylamine; Tyr: tyrosine; Ura: uracil; Val: valine.
5
0
7
4
PAG
PG
PG
IS
7.5
IS
PAG
HA
Cre
HA
3
Cre
PS
PG
2
1
ppm
0.7
Correlation Coefficients (a.u.)
3
Asp
6
8
Ura
Ura
4
PT
MA
2
PT
1
Urine
Feces
S5. O-PLS regression loadings plot shows the correlation between the combination
of urinary and faecal NMR spectral data and Enterobacter hormaechei (Q2Y=0.7;
R2X=28%) level.
6
A
0.45
Urine
HA
PAG
PS AV
HA
PS PG
Cre
Cre
7
7.4
7.8
AV
PG
PAG
Correlation Coefficients (a.u.)
Suc
4
2
2.5
3
3.5
0
1 ppm
1.5
0.5
Asp
Feces
Correlation Coefficients (a.u.)
Asp
PT
MA
PT
Ura
Ura
7.6
6.8
5.8
4
4.5
3.5
3
2
2.5
0
1 ppm
1.5
B
C lostridiaceae(%)
18
80
14
70
12
60
10
50
8
40
6
30
4
20
2
10
0
0
200
250
300
350
400
Enterobacteriaceae(%)
90
16
450
500
0.05
4
0.04
3
0.03
2
0.02
200
250
300
350
400
450
0
500
0.01
200
250
Lachnospiraceae(%)
45
0.6
300
350
400
450
500
0
P as teurellaceae(%)
0.9
0.8
16
35
0.7
14
0.6
12
25
0.5
10
0.3
20
0.4
8
15
0.3
6
10
0.2
4
5
0.1
0.1
0
200
250
300
350
400
450
500
0
200
250
300
P revotellaceae(%)
40
50
45
35
40
450
500
R uminococcaceae(%)
15
250
300
350
400
body weight (g)
450
500
0
500
300
350
400
450
500
200
250
300
350
400
450
500
body weight (g)
Veillonellaceae(%)
2
1
10
5
5
450
1.5
15
10
400
2.5
25
20
20
350
2
250
3
30
25
200
200
body weight (g)
35
30
0
400
body weight (g)
body weight (g)
45
350
300
P orphyromonadaceae(%)
18
40
30
0.2
250
body weight (g)
0.4
0.5
200
body weight (g)
body weight (g)
Incertae Sedis XIII(%)
Micrococcaceae(%)
0.06
5
1
body weight (g)
0.7
Lactobacillaceae(%)
6
0.5
200
250
300
350
400
body weight (g)
450
500
0
200
250
300
350
400
450
500
body weight (g)
S6. (A) O-PLS regression coefficient plot derived from 1H NMR urinary (Q2Y=0.58;
R2X=31.4%) and faecal (Q2Y=0.61; R2X=28%) spectral data against body weight. (B)
7
Scatter plots of bacterial family levels and body weight of rats (red: RYGB-operated;
blue: sham control) at week 2 (circles) and 8 (stars).
p = 0 .0 1
Aerococcaceae
Alcaligenaceae
Alteromonadaceae
Bacillaceae
Bacteroidaceae
Bifidobacteriaceae
Carnobacteriaceae
Clostridiaceae
Coriobacteriaceae
Corynebacteriaceae
Deferribacteraceae
Desulfovibrionaceae
Enterobacteriaceae
Enterococcaceae
Erysipelotrichaceae
Eubacteriaceae
Incertae Sedis XII
Incertae Sedis XIII
Lachnospiraceae
Lactobacillaceae
Methylobacteriaceae
Methylococcaceae
Microbacteriaceae
Micrococcaceae
Moraxellaceae
Pasteurellaceae
Peptococcaceae
Peptostreptococcaceae
Planococcaceae
Porphyromonadaceae
Prevotellaceae
Pseudomonadaceae
Rikenellaceae
Ruminococcaceae
Staphylococcaceae
Streptococcaceae
Veillonellaceae
0. 86
0. 69
0. 52
0. 35
0. 18
0. 01
-0. 15
-0. 32
-0. 49
-0. 66
PG
PAG
creatine
PS
methylamine putrescine
uracil
S7. Cross correlation plots between selected urinary and faecal metabolites and the
abundance levels of 37 bacterial families across matched samples (correlations
significant at p < 0.01) Key: PS, p-cresyl sulfate; PG, p-cresyl glucuronide; PAG,
phenylacetylglycine.
8
Table S1 454 primers use to amplify the V1-V3 regions of the 16S rRNA gene, the
portion in bold is the unique barcode (5mer) that distinguished each sample and
allows for multiplexing of the sample on the 454. To the right of the barcode is the
target primer which anneals to the 16S rRNA gene.
Sample
S1
S2
S3
S4
S5
S6
S7
S8
S9
S10
S11
S12
B1
B2
B3
B4
B5
B6
B7
B8
B9
B10
B11
B12
Forward primer and barcode in bold
CCATCTCATCCCTGCGTGTCTCCGACTCAG GATCT GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG ATCAG GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG ACACT GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG AGCTA GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CACAC GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG ACAGA GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG AGATG GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CACTG GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CAGAG GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CGCAG GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CTGTG GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG GTGAG GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG TCATG GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG AGCAT GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CAGCT GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CATGT GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CTGAT GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CTGCA GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG GATGA GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG TACGC GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG ACTGC GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG GTCAC GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG CGTAC GCCTAACACATGCAAGTC
CCATCTCATCCCTGCGTGTCTCCGACTCAG TGCGT GCCTAACACATGCAAGTC
Reverse primer
CCTATCCCCTGTGTGCCTTGGCAGTCTCAG ATTACCGCGGCTGCTGG
9