SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTARY INFORMATION ARTICLE NUMBER: DOI: /NMICROBIOL A reference gene catalogue of the pig gut microbiome Liang Xiao 1, Jordi Estellé 2, Pia Kiilerich 3, Yuliaxis Ramayo-Caldas 2, Zhongkui Xia 1, Qiang Feng 1, Suisha Liang 1, Anni Øyan Pedersen 4, Niels Jørgen Kjeldsen 4, Chuan Liu 1,5, Emmanuelle Maguin 6, Joël Doré 6,7, Nicolas Pons 7, Emmanuelle Le Chatelier 7, Edi Prifti 7,, Junhua Li 1,8, Huijue Jia 1, Xin Liu 1, Xun Xu 1, Stanislav D. Ehrlich 7,9, Lise Madsen 1,3,10, Karsten Kristiansen 1,3*, Claire Rogel-Gaillard 2* and Jun Wang 1,3* Supplementary information NATURE MICROBIOLOGY Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

2 a Superkingdom (50.8%) Phylum (41.2%) Genera (7.6%) Species (0.33%) 3,782,287 3,903, ,465 3,170, , , ,792 25,336 Gene Annotation Superkingdom Phylum Genus Species Bacteria Archaea Eukaryota Firmicutes Proteobacteria Actinobacteria Fibrobacteres Other Bacteroidetes Spirochaetes Euryarchaeota Verrucomicrobia Prevotella Clostridium Eubacterium Lactobacillus Bacteroides Ruminococcus Butyrivibrio Other Escherichia coli Faecalibacterium prausnitzii Methanobrevibacter smithii Ruminococcus obeum Phascolarctobacterium succinatutens Prevotella copri Acidaminococcus fermentans Dorea longicatena Lactobacillus amylovorus Other b Superkingdom (69.1%) Phylum Genera Species (49.1%) (4.6%) (0.14%) MGS Annotation Superkingdom Phylum Genus Species Bacteria Archaea Firmicutes Proteobacteria Fibrobacteres Bacteroidetes Euryarchaeota Prevotella Eubacterium Lactobacillus Dorea Clostridium Butyrivibrio Streptococcus Ruminococcus Methanobrevibacter Helicobacter Coprococcus Catenibacterium Acidaminococcus Phascolarctobacterium Escherichia coli Supplementary Figure 1 Supplementary Figure 1 Distribution of microbial gene (a) and MGS (b) annotated against the NR database. Only annotated genes/mgs (green areas in panels a & b) were included in the analysis. 50.8% of the NR genes and 69.1% of the MGS could be annotated to the Bacteria super-kingdom, demonstrating that many unknown genes are detected in the pig gut microbiota. Further taxonomic annotation was carried out for the NR genes assigned to Bacteria and Archae, with 41.2 and 7.6% genes annotated at the phylum and genus levels, respectively. Less than 1% of the genes could be annotated at the species level (0.33%).

3 a Distribution of KOs from annotated species Organismal Systems Sensory System Organismal Systems Nervous System Organismal Systems Immune System Organismal Systems Excretory System Organismal Systems Environmental Adaptation Organismal Systems Endocrine System Organismal Systems Digestive System Organismal Systems Development Organismal Systems Circulatory System Metabolism Xenobiotics Biodegradation and Metabolism Metabolism Nucleotide Metabolism Metabolism Metabolism of Terpenoids and Polyketides Metabolism Metabolism of Other Amino Acids Metabolism Metabolism of Cofactors and Vitamins Metabolism Lipid Metabolism Metabolism Glycan Biosynthesis and Metabolism Metabolism Enzyme Families Metabolism Energy Metabolism Metabolism Carbohydrate Metabolism Metabolism Biosynthesis of Other Secondary Metabolites Metabolism Amino Acid Metabolism Human Diseases Neurodegenerative Diseases Human Diseases Metabolic Diseases Human Diseases Infectious Diseases Human Diseases Immune System Diseases Human Diseases Cardiovascular Diseases Human Diseases Cancers Genetic Information Processing Translation Genetic Information Processing Transcription Genetic Information Processing Replication and Repair Genetic Information Processing Folding, Sorting and Degradation Environmental Information Processing Signaling Molecules and Interaction Environmental Information Processing Signal Transduction Environmental Information Processing Membrane Transport Cellular Processes Transport and Catabolism Cellular Processes Cell Motility Cellular Processes Cell Growth and Death Cellular Processes Cell Communication Distribution of KOs from MGS 5e+05 4e+05 3e+05 2e+05 1e+05 0e+00 0e+00 1e+05 2e+05 3e+05 4e+05 5e+05 b Distribution of COGs from annotated species U : Intracellular trafficking, secretion, and vesicular transport G : Carbohydrate transport and metabolism I : Lipid transport and metabolism R : General function prediction only D : Cell cycle control, cell division, chromosome partitioning H : Coenzyme transport and metabolism B : Chromatin structure and dynamics P : Inorganic ion transport and metabolism W : Extracellular structures J : Translation, ribosomal structure and biogenesis O : Posttranslational modification, protein turnover, chaperones A : RNA processing and modification L : Replication, recombination and repair C : Energy production and conversion M : Cell wall/membrane/envelope biogenesis Q : Secondary metabolites biosynthesis, transport and catabolism : Z : Cytoskeleton V : Defense mechanisms E : Amino acid transport and metabolism Y : Nuclear structure K : Transcription N : Cell motility T : Signal transduction mechanisms S : Function unknown F : Nucleotide transport and metabolism Distribution of COGs from MGS 4e+05 3e+05 2e+05 1e+05 0e+00 0e+00 1e+05 2e+05 3e+05 4e+05 Supplementary Figure 2 Supplementary Figure 2 KEGG (a) and eggnog (b) annotation of the NR genes assigned to bacterial species (left hand) and annotation of the NR genes clustered into MGS (right hand). The functional annotation based on all NR genes and on the genes clustered in the MGS was found to be consistent. COG: Cluster of Orthologous Groups.

4 Distribution of KOs from annotated species Distribution of KOs shared by all the pig samples Organismal Systems Sensory System Organismal Systems Nervous System Organismal Systems Immune System Organismal Systems Excretory System Organismal Systems Environmental Adaptation Organismal Systems Endocrine System Organismal Systems Digestive System Organismal Systems Development Organismal Systems Circulatory System Metabolism Xenobiotics Biodegradation and Metabolism Metabolism Nucleotide Metabolism Metabolism Metabolism of Terpenoids and Polyketides Metabolism Metabolism of Other Amino Acids Metabolism Metabolism of Cofactors and Vitamins Metabolism Lipid Metabolism Metabolism Glycan Biosynthesis and Metabolism Metabolism Enzyme Families Metabolism Energy Metabolism Metabolism Carbohydrate Metabolism Metabolism Biosynthesis of Other Secondary Metabolites Metabolism Amino Acid Metabolism Human Diseases Neurodegenerative Diseases Human Diseases Metabolic Diseases Human Diseases Infectious Diseases Human Diseases Immune System Diseases Human Diseases Cardiovascular Diseases Human Diseases Cancers Genetic Information Processing Translation Genetic Information Processing Transcription Genetic Information Processing Replication and Repair Genetic Information Processing Folding, Sorting and Degradation Environmental Information Processing Signaling Molecules and Interaction Environmental Information Processing Signal Transduction Environmental Environmental Information Processing Membrane Transport Cellular Processes Transport and Catabolism Cellular Processes Cell Motility Cellular Processes Cell Growth and Death Cellular Processes Cell Communication 7e+05 6e+05 5e+05 4e+05 3e+05 2e+05 1e+05 0e+00 0e+00 1e+05 2e+05 3e+05 4e+05 5e+05 6e+05 7e+05 Supplementary Figure 3 Supplementary Figure 3 KEGG annotation of gut microbiome biological functions that are 100% shared by the whole set of 287 pigs. The shared functions are consistent with those identified for the entire pig gut gene catalogue both in relation to annotation and relative abundances.

5 a b Supplementary Figure 4 Supplementary Figure 4 Functional comparison of the pig, human and mouse catalogues. The Venn diagram (a) provides the number of shared and species-specific KEGG pathways for the pig (pink), the mouse (grey), and the human (yellow) catalogues. The classification of the 2179 KEGG pathways found 100% shared by the three animal species (b) highlights the predominance of common metabolic functions related to carbohydrates and amino acids as well as environmental information processing (membrane transport), consistent with the most abundant functions found in the pig catalogue (see Supplementary Fig. 2 and Supplementary Fig. 3).

6 a b c d e Supplementary Figure 5 Supplementary Figure 5 Effects of host genetics on the pig microbiome composition. The influence of host genetics was assessed by NMDS from the subset of Chinese pigs, at the levels of phylum (a), genus (b), species (c), MGS (d) and KEGG pathways (e). The MGS-based NMDS clearly distinguished three groups corresponding to the highly selected commercial breed (HybCN1, HybCN2 and Large White), the Bama and related BaRing pigs, and the Tibetan pigs. The KEGG-based NMDS still clearly separated the Tibetan pigs from the others, suggesting specific microbiota functions in this breed.

7 a b c Supplementary Figure 6 Supplementary Figure 6 Age effect on the pig gut microbiota composition. The influence of age was assessed by NMDS at the levels of the total NR gene counts (a) and KEGG pathways (b, c), from the subsets of Danish pigs (a, c) and French pigs (b) (counterpart of Fig. 3b in the main text). The animals are distributed in a surface plot that includes lines referring to their ages, showing an effect of age, likely to be connected to the diet and environmental changes during lifetime.

8 a b c Supplementary F igure 7 Supplementary Figure 7 Effect of the farm system (country) on the gut microbiota composition. NMDS based on the NR gene counts (a), the KEGG pathways (b) and the MGS counts revealed a separation between the Chinese pigs CP), and the French (FP) and Danish (DP) pigs. This separation is likely to be partly related to different farm systems. In addition, the French pigs comprised 11 subsets divided by the breed, the environment, or both, resulting in more diversity than for the Danish pigs, as revealed by this analysis.

9 a Aminoglycosides d b Beta-lactams c Sulfonamide Supplementary Figure 8 Supplementary Figure 8 Country-specific relative abundances of subsets of antibiotic resistance genes (ARGs) and of KOs related to the tricarboxylic acid (TCA) cycle. (a) Aminoglycosides; (b) Beta-lactams; (c) Sulfonamide; (d) KOs in the TCA cycle.

10 Supplementary table 1. Background information on the 287 pig samples. Supplementary table 2. Description of the assembly data from the 287 samples. Supplementary table 3. Assembly results of the pig, human and mouse data. Supplementary table 4. Significant differences in abundance between the gut microbiomes of castrated males and females (10 castrated males, 10 females, Svindinge farm) at the genus and species levels. Supplementary table 5. Significant differences in abundance between the gut microbiomes of castrated males and females (10 castrated males, 10 females, Svindinge farm) at the KEGG pathway level. Supplementary table 6. Significant differences in abundance between the gut microbiomes of males and females (11 males, 14 females, fed wet feed, Stærsminde farm) at the genus and species levels. Supplementary table 7. Significant differences in the abundance of MGS in the gut microbiomes of males and females (11 males, 14 females, fed wet feed, Stærsminde farm). Supplementary table 8. Significant differences in KEGG pathways in the gut microbiota of males and females (11 males, 14 females, fed wet feed, Stærsminde farm). Supplementary table 9. List of the KEGG pathways mapped by ipath2 that differed significantly between the gut microbiota of males and females (11 males, 14 females, fed wet feed, Stærsminde farm). The three most represented pathways are highlighted in grey. This list is derived from table S8 and corresponds to the differentially abundant functions mapping against KEGG according to ipath2 tools.