© 2016 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

A reference gene catalogue of the pig gut microbiome

LiangXiao1†,JordiEstellé2†,PiaKiilerich3†,YuliaxisRamayo-Caldas2,ZhongkuiXia1,Qiang

Feng1‡,SuishaLiang1,AnniØyanPedersen4,NielsJørgenKjeldsen4,ChuanLiu1,5,Emmanuelle

Maguin6,JoëlDoré6,7,NicolasPons7,EmmanuelleLeChatelier7,EdiPrifti7,§,JunhuaLi1,8,

HuijueJia1,XinLiu1,XunXu1,StanislavD.Ehrlich7,9,LiseMadsen1,3,10,KarstenKristiansen1,3*,

ClaireRogel-Gaillard2*andJunWang1,3*

Supplementary information

SUPPLEMENTARY INFORMATIONARTICLE NUMBER: 16161 | DOI: 10.1038/NMICROBIOL.2016.161

NATURE MICROBIOLOGY | www.nature.com/naturemicrobiology 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%).

Genus

Prevotella Bacteroides Clostridium Ruminococcus Eubacterium Butyrivibrio Lactobacillus Other Unknown

Species

Escherichia coli

Methanobrevibacter smithii

Faecalibacterium prausnitzii

Ruminococcus obeum

Phascolarctobacterium succinatutens Prevotella copri

Acidaminococcus fermentans Dorea longicatena

Lactobacillus amylovorus Other

Unknown

Superkingdom(50.8%)

Phylum(41.2%)

Genera(7.6%)

Species(0.33%)

3,903,5853,782,287 3,170,194689,465587,128444,626 25,336

561,792

Superkingdom

Bacteria Archaea UnknownFirmicutes Bacteroidetes

Proteobacteria Euryarchaeota

Fibrobacteres Unknown

Prevotella Eubacterium Lactobacillus Dorea Clostridium Butyrivibrio

Ruminococcus Methanobrevibacter Helicobacter Coprococcus Catenibacterium AcidaminococcusPhascolarctobacteriumStreptococcus

Unknown

Escherichia coli

Unknown

Phylum Genera Species(69.1%)

497222

(49.1%)

353144

(4.6%)

3312 132

(0.14%)

Gene Annotation

MGS Annotation

a

b

Superkingdom Unknown Phylum Unknown Unknown Unknown

BacteriaArchaea

Eukaryota

Bacteroidetes Spirochaetes Euryarchaeota Verrucomicrobia Unknown

Firmicutes Proteobacteria Actinobacteria Fibrobacteres Other

Superkingdom Unknown Phylum Unknown Genus Unknown Species Unknown

Supplementary Figure 1

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.

P : Inorganic ion transport and metabolism

a

b

Supplementary Figure 2

Fu

nc

tio

n c

las

s

Number of matched genes

Cellular Processes −− Cell CommunicationCellular Processes −− Cell Growth and Death

Cellular Processes −− Cell MotilityCellular Processes −− Transport and Catabolism

Environmental Information Processing −− Membrane TransportEnvironmental Information Processing −− Signal Transduction

Environmental Information Processing −− Signaling Molecules and InteractionGenetic Information Processing −− Folding, Sorting and Degradation

Genetic Information Processing −− Replication and RepairGenetic Information Processing −− Transcription

Genetic Information Processing −− TranslationHuman Diseases −− Cancers

Human Diseases −− Cardiovascular DiseasesHuman Diseases −− Immune System Diseases

Human Diseases −− Infectious DiseasesHuman Diseases −− Metabolic Diseases

Human Diseases −− Neurodegenerative DiseasesMetabolism −− Amino Acid Metabolism

Metabolism −− Biosynthesis of Other Secondary MetabolitesMetabolism −− Carbohydrate Metabolism

Metabolism −− Energy MetabolismMetabolism −− Enzyme Families

Metabolism −− Glycan Biosynthesis and MetabolismMetabolism −− Lipid Metabolism

Metabolism −− Metabolism of Cofactors and VitaminsMetabolism −− Metabolism of Other Amino Acids

Metabolism −− Metabolism of Terpenoids and PolyketidesMetabolism −− Nucleotide Metabolism

Metabolism −− Xenobiotics Biodegradation and MetabolismOrganismal Systems −− Circulatory System

Organismal Systems −− DevelopmentOrganismal Systems −− Digestive System

Organismal Systems −− Endocrine SystemOrganismal Systems −− Environmental Adaptation

Organismal Systems −− Excretory SystemOrganismal Systems −− Immune SystemOrganismal Systems −− Nervous SystemOrganismal Systems −− Sensory System

0e+00 1e+05 2e+05 3e+05 4e+05 5e+05

Distribution of KOs from MGS

Fu

nc

tio

n c

las

s

Number of matched genes0e+001e+052e+053e+054e+055e+05

Distribution of KOs from annotated species

Distribution of COGs from MGSDistribution of COGs from annotated species

Fu

ncti

on

cla

ss

Number of matched genes

F : Nucleotide transport and metabolismS : Function unknown

T : Signal transduction mechanismsN : Cell motility

K : TranscriptionY : Nuclear structure

E : Amino acid transport and metabolismV : Defense mechanisms

Z : Cytoskeleton− : Unknown

Q : Secondary metabolites biosynthesis, transport and catabolismM : Cell wall/membrane/envelope biogenesis

C : Energy production and conversionL : Replication, recombination and repair

A : RNA processing and modificationO : Posttranslational modification, protein turnover, chaperones

J : Translation, ribosomal structure and biogenesisW : Extracellular structures

B : Chromatin structure and dynamicsH : Coenzyme transport and metabolism

D : Cell cycle control, cell division, chromosome partitioningR : General function prediction only

I : Lipid transport and metabolismG : Carbohydrate transport and metabolism

U : Intracellular trafficking, secretion, and vesicular transport

0e+00 1e+05 2e+05 3e+05 4e+05

Fu

ncti

on

cla

ss

Number of matched genes0e+001e+052e+053e+054e+05

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.

Distribution of KOs shared by all the pig samples

Function c

lass

Organismal Systems −− Sensory SystemOrganismal Systems −− Nervous SystemOrganismal Systems −− Immune System

Organismal Systems −− Excretory SystemOrganismal Systems −− Environmental Adaptation

Organismal Systems −− Endocrine SystemOrganismal Systems −− Digestive System

Organismal Systems −− DevelopmentOrganismal Systems −− Circulatory System

Metabolism −− Xenobiotics Biodegradation and MetabolismMetabolism −− Nucleotide Metabolism

Metabolism −− Metabolism of Terpenoids and PolyketidesMetabolism −− Metabolism of Other Amino Acids

Metabolism −− Metabolism of Cofactors and VitaminsMetabolism −− Lipid Metabolism

Metabolism −− Glycan Biosynthesis and MetabolismMetabolism −− Enzyme Families

Metabolism −− Energy MetabolismMetabolism −− Carbohydrate Metabolism

Metabolism −− Biosynthesis of Other Secondary MetabolitesMetabolism −− Amino Acid Metabolism

Human Diseases −− Neurodegenerative DiseasesHuman Diseases −− Metabolic DiseasesHuman Diseases −− Infectious Diseases

Human Diseases −− Immune System DiseasesHuman Diseases −− Cardiovascular Diseases

Human Diseases −− CancersGenetic Information Processing −− Translation

Genetic Information Processing −− TranscriptionGenetic 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 TransportCellular Processes −− Transport and Catabolism

Cellular Processes −− Cell MotilityCellular Processes −− Cell Growth and Death

Cellular Processes −− Cell CommunicationF

unction c

lass

Distribution of KOs from annotated species

0e+001e+055e+05 4e+05 3e+05 Number of matched genes

2e+056e+057e+05 0e+00 1e+05 2e+05 3e+05 4e+05 Number of matched genes

5e+05 6e+05 7e+05

Supplementary Figure 3

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).

a

b

Supplementary Figure 4

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.

a b c

d e

Supplementary Figure 5

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.

Supplementary Figure 6

a b c

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.

Supplementary F igure 7

c

a b

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.

Beta-lactams

Aminoglycosides

Sulfonamide

A

B

C

D a Aminoglycosides

b Beta-lactams

c Sulfonamide

d

Supplementary Figure 8

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.