A.

B.

Proteobacteria

Proteobacteria

Epsilon Proteobacteria

Epsilon Proteobacteria

Supplemental Figure 1. Consensus sequences for (A) forward and (B) reverse primers based on alignment of 49 proteobacteria (upper panels) or a subset of 19 Epsilon proteobactiera (lower panels). Sequences of the Epsilon enrichment primers JH0108 and JH0102 are shown below the Epsilon proteobacteria consensus sequences. Figures were generated by Weblogo (http://weblogo.berkeley.edu/).

5'-ATGAANTTTCARCCWYTWGG-3’ JH0108

5'-ARCATHKCTTTTCTTCTRTC-3’ JH0102

Campylobacter jejuni (ε) Campylobacter fetus (ε) Helicobacter sp. (ε)

Salmonella (γ) Pasteurella (γ)

Pseudomonas (γ) Bartonella (α)

Supplemental Figure 2. Temperature gradient PCR with Epsilon-proteobacteria enrichment primers on pure proteobacteria cultures. PCR primers JH0108 and JH0102 were tested with genomic DNA extracts in a gradient PCR with annealing temperatures from 49.3°C to 60.1°C (indicated by increasing triangle), plus no template control (-). Arrows indicate the 1200 bp marker . The cpn10-cpn60 target of ~1200 bp was clearly seen with epsilon-proteobacteria genomic DNA (C. jejuni, C. fetus and Helicobacter sp.), while only non-specific bands were obtained from gamma- and alpha-proteobacteria genomic DNA (Salmonella, Pasteurella, Pseudomonas and Bartonella samples).

Supplemental Figure 3. Rarefaction curves of dog fecal microbiota libraries generated in this study. Both rarefaction analysis and the Good’s coverage estimate (given in parentheses after each library) were calculated using mothur software.

Supplemental figure 3 (continued). Rarefaction curves of dog fecal microbiota libraries generated in this study. Both rarefaction analysis and the Good’s coverage estimate (given in parentheses after each library) were calculated using mothur software.

Supplemental Figure 4A. A jackknifed clustering of the samples by Unifrac distances (100 permutations) of non-enriched libraries. The numbers at the nodes indicate the number of times that particular node was observed (out of 100) in a random sampling of the dataset. Brackets highlight the distinction between the cluster that is predominantly healthy dog libraries and the cluster that is predominantly diarrheic dog libraries.

0.1

HDS1fall

HDS2fall

HDS2spring

DDS18

HDS8

HDS1spring

HDS18D

HDS30

DDS2

DDS64

DDS34

HDS19

DDS19

DDS32

DDS30

HDS9A

DDS11

DDS51

43

70

87

100

100

61

70

87

84

100

100

100

100

100

49

100

0.1

HDS19.enrich

HDS1fall.enrich.rep

HDS1fall.enrich

DDS19.enrich

DDS30.enrich

HDS2fall.enrich

HDS2spring.enrich

HDS18D.enrich

HDS1spring.enrich

HDS8.enrich

HDS9A.enrich

DDS51.enrich

HDS30.enrich

DDS34.enrich

DDS2.enrich

DDS32.enrich

DDS64.enrich

DDS18.enrich

HDS9A.universal

HDS1fall.universal

HDS2fall.universal

HDS2spring.universal

DDS18.universal

HDS8.universal

HDS1spring.universal

HDS18D.universal

HDS30.universal

DDS19.universal

DDS32.universal

DDS2.universal

DDS64.universal

DDS34.universal

HDS19.universal

DDS11.universal

DDS51.universal

DDS11.enrich

DDS30.universal

100100

10098

100

100

97

100100

97

97

97

85

100

72

62

65

67

36

34

42

67

48

71

97

10099

100

100

98

100

100100

100100

Supplemental Figure 4B. A jackknifed clustering of the samples by Unifrac distances (100 permutations) of all libraries generated in the study. The numbers at the nodes indicate the number of times that particular node was observed (out of 100) in a random sampling of the dataset. Brackets highlight the distinction between the cluster that is predominantly universal (non-enriched) cpn60 libraries and the cluster that is predominantly epsilon-proteobacteria enriched libraries.

PhylumHDS1fall_enrich1

HDS1fall_enrich2

Actinobacteria <1a 22 Bacteroidetes 4 2

Firmicutes 531 183 Proteobacteria 2466 2796a Scaled number of reads in library

GenusHDS1fall_enrich1

HDS1fall_enrich2

Aeromonas 0a 10 Atopobium <1 7 Bacteroides 4 2

Bifidobacterium 0 5 Blautia 1 0

Butyrivibrio 270 51 Campylobacter 34 0

Clostridium 1 0 Coprococcus 252 29 Cupriavidus 0 5Enterobacter <1 0 Gardnerella 0 10

Hafnia 0 2 Helicobacter 2429 2659 Lactobacillus 1 49

Peptostreptococcus <1 39 Ruminococcus 4 0

Shewanella 2 120 Streptococcus <1 15

a Scaled number of reads in library

Supplemental Figure 5. Comparison of technical replicates of HDS1fall at the (A) phylum and (B) genus level.

A

B

126 isotigs (possible novel Helicobacter sp.)isotig 232

12 isotigs

H. cinaediisotig 536

22 isotigs

H. canis

H. hepaticus

isotig 164isotig 646

H. mustelae

H. nemestrinae

H. acinonychis

H. fennelliae

isotig 887 (Flexispira rappini OTU)

isotig 864

H. felisH. salomonis

H. bizzozeroniiH. pylori

H. cholecystus

H. winghamensisH. pametensis

H. muridarumH. pullorum

H. bilis

H. trogontum

isotig 027

isotig 731A. skirrowii

H. canadensis

A. nitrofigilis

C. sputorum

isotig 905

isotig 579

isotig 883

A. mytili

C. hyointestinalis

C. upsaliensis

C. helveticusC. coli

C. fetus venerealis

C. jejuni

C. fetus fetus

C. gracilis

C. retus

C. lari

isotig 585isotig 632 (possible novel Campylobacter species)

isotig 594

C. showae

C. concisusC. mucosalis

C. curvusE. coli

0.1

Supplemental Figure 6. Phylogenetic tree of epsilon-proteobacteria OTU. A 100 bp region (from nucleotide 200-300 within the cpn60 UT region) was aligned and used to generate a neighbour-joined tree. Alignments were manually inspected to identify any anomolous sequences that incorrectly passed quality control (none were identified). Species are highlighted by coloured backgrounds. Isotigs (equivelant to OTU) that represent possible novel species have been highlighted with the label “(possible novel X species)”.