Bacteria Proteobacteria Purple phototrophic bacteria Bacteriochlorophyll Carotenoids.
A. B. Proteobacteria Epsilon Proteobacteria Epsilon Proteobacteria Supplemental Figure 1. Consensus...
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Transcript of A. B. Proteobacteria Epsilon Proteobacteria Epsilon Proteobacteria Supplemental Figure 1. Consensus...
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.
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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
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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
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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)”.