Super-organism. 2 kg microbes microbial cells (10 12 human cells) 10M microbial genes (25000 human genes)
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1 Microbiology and NGS Henrik Bjørn Nielsen Center for Biological Sequence Analysis, Department of Systems Biology, Technical University of Denmark
2 Microbiome
3 Super-organism 2 kg microbes microbial cells (10 12 human cells) 10M microbial genes (25000 human genes)
4 Human Microbiota Phylums of Animalia
5 Microbiota and obesity Microbiota transplantation from obese or lean mice into sterile mice Bäckhed et al (PNAS)
6 Motivation
7 1 Citrobacter sp. 30_2 2 Actinomyces odontolyticus 3 Enterobacter sp Streptococcus mutans 5 Lactobacillus gasseri 6 Pasteurella multocida 7 Pediococcus pentosaceus 8 Lactobacillus helveticus 9 Campylobacter concisus 10 Enterobacter cancerogenus 11 Lactobacillus reuteri 12 Lactobacillus sakei 13 Streptococcus suis 14 Streptococcus thermophilus 15 Actinobacillus pleuropneumoniae 16 Haemophilus parasuis 17 Cronobacter sakazakii 18 Haemophilus influenzae 19 Lactobacillus salivarius Streptococcus gordonii 21 Lactobacillus johnsonii 22 Lactobacillus fermentum 23 Citrobacter sp. 24 Escherichia fergusonii 25 Streptococcus pneumoniae 26 Streptococcus pyogenes 27 Salmonella enterica 28 Citrobacter koseri 29 Escherichia coli 30 Lactobacillus hilgardii 31 Blautia hansenii 32 Clostridium scindens 33 Streptococcus sanguinis 34 Clostridium perfringens 35 Lactobacillus ultunensis Klebsiella pneumoniae Actinobacteria Bacteroidetes Firmicutes Fusobacteria Proteobacter Tenericutes Streptococcus infantarius Eubacterium dolichum Anaerostipes caccae Ruminococcus gnavus Clostridium leptum Clostridium ramosum Clostridium bolteae Faecalibacterium prausnitzii Mollicutes bacterium D7 Positive correlation Negative correlation Holdemania filiformis Bifidobacterium pseudocatenulatum Clostridium nexile Anaerotruncus colihominis Leuconostoc mesenteroides Gordonibacter pamelaeae Clostridium asparagiforme Bifidobacterium dentium Bacteroides capillosus Subdoligranulum variabile Clostridium symbiosum Thermoanaerobacter sp. X514 Collinsella stercoris Lactobacillus paracasei Bifidobacterium gallicum Clostridium methylpentosum Dorea longicatena Bifidobacterium breve Collinsella aerofaciens Bifidobacterium longum Bifidobacterium adolescentis Bacteroides sp. 2_2_4 Staphylococcus saprophyticus Bifidobacterium animalis Ruminococcus obeum Dorea formicigenerans Coprococcus comes Eubacterium hallii Bifidobacterium bifidum Bacteroides xylanisolvens Bacteroides sp. D1 Bacteroides uniformis Bacteroides vulgatus Ruminococcus sp. Eubacterium ventriosum Clostridium sp Bacteroides ovatus Bacteroides sp. 9_1_42FAA Bacteroides sp. D4 Bacteroides dorei Bacteroides sp. 4_3_47FAA Bifidobacterium catenulatum Lactobacillus acidophilus Enterococcus faecalis Bryantella formatexigens Lactobacillus casei Clostridium phytofermentans Bacteroides fragilis Candidatus Sulcia muelleri Finegoldia magna Clostridium hylemonae Tropheryma whipplei Butyrivibrio fibrisolvens Lactobacillus delbrueckii Anaerofustis stercorihominis Proteus penneri Desulfovibrio vulgaris Megamonas hypermegale Porphyromonas gingivalis Clostridium difficile Mitsuokella multacida Proteus mirabilis Clostridium bartlettii Blautia hydrogenotrophica Anaerococcus hydrogenalis Lactococcus lactis Fusobacterium nucleatum Clostridium sp. 7_2_43FAA Desulfovibrio piger Qin et al. (Nature 2010)
8 Groups of ecosystems Enterotypes!
9 RESEARCH ARTICLE Enterotypes a b JP.AD.7 Bacteroides JP.AD.1 JP.AD.9 FR.AD.3 JP.AD.8 JP.AD.6 ES.AD.1 JP.AD.4 Roseburia Catenibacterium *Lachnospiraceae Subdoligranulum Mitsuokella *Peptostreptococcaceae Bacteroides Prevotella IT.AD.4 Prevotella ES.AD.3 ES.AD.2 DA.AD.1 JP.AD.2 PC1 FR.AD.7 ES.AD.4 IT.AD.3 FR.AD.2 JP.AD.3 JP.AD.5 FR.AD.8 Akkermansia AM.F10.T2 FR.AD.5 AM.F10.T1 IT.AD.1 Alistipes FR.AD.1 IT.AD.6 DA.AD.3 Ruminococcus DA.AD.2 c Bacteroides Blautia DA.AD.4 FR.AD.6 FR.AD.4 Obese IT.AD.5 IT.AD.2 Prevotella IBD PC2 d Bacteroides Prevotella Ruminococcus Abundance Enterotype e Alkaliphilus Geobacter Catenibacterium Parabacteroides Bacteroides Clostridiales Lactobacillus Methanobrevibacter Slackia Enterotype Enterotype Veillonella Akkermansia Akkermansia Helicobacter Sphingo Gordonibacter *Ruminococcaceae Leuconostoc bacterium Ruminococcus Staphylococcus *Rumino Eggerthella coccaceae Prevotella *Peptostrepto Staphylococcus coccaceae Desulfovibrio Holdemania Marvinbryantia Dialister Rhodospirillum Symbiobacterium Escherichia/Shigella Main contributors Genera co-occurring with main contributors Positive correlation (>0.4) Negative correlation (< 0.4) Figure 2 Phylogenetic differences between enterotypes. a c, Between-class analysis, which visualizes results from PCA and clustering, of the genus compositions of 33 Sanger metagenomes estimated by mapping the R with each sample represented by a filled circle. The centre of gravity for each cluster is marked by a rectangle and the coloured ellipse covers 67% of the samples belonging to the cluster. IBD, inflammatory bowel disease. Arumugam et al. (Nature, 2011)
10 microbial communities Differentiation Dynamic Competition Mutualism Richness Antibiotic resistance
11 * insulin resistance, adiposity, dyslipidaemia and inflammation markers (Chatelier et al. nature, 2013) Richness of the human gut metagenomic species per sample Crohn s Crohn s disease: Enterotype: R P B Metabolic markers and inflammation*
12 Richness of the gut microbiome Fang and Evans., Nature 2013 Le Chatelier et al., Nature 2013 Cotillard et al., Nature 2013
13 Persistence Longitudinal sampling Kaplan-Meyer estimator Time
14 Rarefaction curve
15 The sampling effect
16 Downsizing To make fair comparisons we should sample equally carefully in all systems Did you find any bacteria? No - not here
17 Downsizing Resample an equal number of observations (reads) from each sample Select the sampling depth carefully The more reads the more sensitive We may have to discharge samples with fewer read counts
SUPPLEMENTARY FIGURES & TABLES
SUPPLEMENTARY FIGURES & TABLES Figures S1-S6 Tables S1-S5 A 6 Bacteroidetes/Firmicutes B 1.0 Prevotellaceae/Bacteroidetes Ratio 5 4 3 2 Ratio 0.8 0.6 0.4 1 0.2 0 1 2 3 4 5 6 BSS 0.0 1 2 3 4 5 6 BSS Supplementary
Supplementary Figure 1
Supplementary Figure 1. (A C) Comparison of Shannon evenness between children who became anti islet autoantibody positive (anti islet aab+) and children who remained autoantibody negative (anti islet aab
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(43) International Publication Date WO 2015/ A2 25 June 2015 ( ) W P O P C T
(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (19) World Intellectual Property Organization International Bureau (10) International Publication Number (43) International
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Received 3 April 2008/Returned for modification 5 May 2008/Accepted 17 October 2008
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bifidobacteria Genus Bacteroides fragilis Non-specific longum, B. breve and B. longum bv. Infantis CGACGATCCCAGAGATGG Bifidobacterium longum and
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