Metagenomic Analysis in Human- Associated Projects
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1 Metagenomic Analysis in Human- Associated Projects Wikimedia Commons Wikimedia Commons Daniel H. Huson Singapore Center for Environmental Life Science Engineering (SCELSE) ZBIT Center for Bioinformatics
2 Overview Metagenomics Adipositas Inflammatory bowel disease Waste water treatment
3 Overview Metagenomics Adipositas Inflammatory bowel disease Waste water treatment
4 Metagenomics " The study of the DNA of uncultured organisms " > 99% of all microbes cannot be cultured " A genome: " EnGre genegc informagon of a single organism " A metagenome: " EnGre genegc informagon of a community of organisms " A metatranscriptome: " EnGre collecgon of transcripts of a community at a given Gme 4
5 Next- Genera=on Sequencing Technologies " Fuelling a rapid growth of the number and size of sequencing projects " Soon: 1 tera base per run 5
6 Center for Bioinformatics Typical Sources of Metagenomes " Soil samples " Sea water samples " Air samples " Ancient bones " Water treatment samples " Medical samples " Human microbiome 6
7 Gut Microbiota " Turnbaugh et al. (2006) " Caecal microbial DNA of ob/ob, ob/+, +/+ mice " Sanger sequencing: " 39.5 Mb " read length 750 bp " 454 sequencing: " 160 Mb " read length 93 bp " Change in relagve abundance of Bacteroidetes and Firmicutes " Change in funcgonal capacity (toward energy harvesgng) 7
8 Large- Scale Human Gut Analysis Taxonomic analysis using MEGAN 576 Gb of sequence from 124 individuals 8
9 Core of Human Gut Microbiome " 57 species present in 90% of individuals with coverage >1% " High variability " Bacteroidetes and Firmicutes most abundant 9
10 Human Microbiome Project " Metagenomic 16S: 50 million reads " Metagenomic shotgun: ~10 11 reads " 3000 reference genomes, including eukaryotes and viruses " Main quesgon: DisGnguish a healthy from a perturbed microbiome? From George Weinstock s talk (2011): What s next for the Human Microbiome? 10
11 Metagenomic Unfolds Hype True performance Level of excitement Enthusiasm Initial idea or here? Disillusionment Are we here Time From George Weinstock s talk (2011): What s next for the Human Microbiome? 11
12 Center for Bioinformatics Metagenome Analysis High-throughput DNA sequencing Environmental sample Basic computational analysis Q1: Who is out there? Q2: What are they doing? 12
13 Q3: How Do They Compare?
14 MEGAN MEtaGenome ANalyzer Huson et al., 2007, 2011 " InteracGve tool for metagenomic analysis www-ab.informatik.uni-tuebingen.de/software/megan 14
15 Iden=fying Taxa and Genes Metagenome analysis Basic idea: compare reads against references sequences of known species and/or function sample DNA Reads Sequence comparison Comparison data Metagenome NCBI-nr NCBI-nt Interactive analysis and visualization Reference databases - Computational analysis requires significant resources - Order of magnitude more expensive than sequencing 15
16 Taxonomic Analysis using MEGAN Based on NCBI taxonomy Data from Turnbaugh et al.,
17 Func=onal Analysis using MEGAN Based on SEED classification Overbeek et al., NAR,
18 KEGG Func=onal Analysis Kanehisa et al., NAR (2010) 18
19 Overview Metagenomics Adipositas Inflammatory bowel disease Waste water treatment
20 Adipositas and Gut Microbiome " Joint project with: " Stephan Bischoff and Karin Föster- Fromme (Uni Hohenheim) " Saskia Biskup (CeGAT, Tübingen) " Suparna Mitra (BioinformaGcs, Tübingen) " Analysis of the intesgnal microbiome in different models of obesity and obesity- associated liver disease BMBF Competence Network Adipositas 20
21 Adipositas and Gut Microbiome " Will sequence 48 gut microbiomes to study the effect of different treatments for Adipositas: " OpGfast 52 diet " Gastric sleeve resecgon " Time points: " before, aker 3 months, aker 6 months " Pilot study: K. Förster- Fromme et al, submived 21
22 Adipositas and Gut Microbiome Human gut metagenome 3.6 mio SOLiD reads 22
23 Time Series Unpublished data 23
24 Overview Metagenomics Adipositas Inflammatory bowel disease Waste water treatment
25 Metagenomics of Gut Microbiota in Enteric Diseases Role of the microbiota in Yersinia infection Role of the microbiota in IBD mouse model Gnotobiotic Mouse model to analyze microbiota and enteric disease BMBF program on Medical Infection Genomics 25
26 Gut Microbiota and IBD " ObjecGve 1: Characterize intesgnal microbiota that trigger inflammatory bowel disease (IBD) in a mouse model " Taxonomic comparison: Conventional (CV) gut microbiome SPF gut microbiome Rag1-/- Rag1-/- " Which bacteria in the CV microbiota are involved in triggering coligs? Julia Frick 26
27 Bacteroidetes prevents induction of colitis CV Rag1 -/- mice E hi B lo mice prone to COLITIS log 10 CFU / g feces Antibiotics + B. vulgatus + T cells log 10 CFU / g feces NO COLITIS Decrease of ratio Enterobacteriaceae to Bacteroidetes: Prevents colitis E lo B hi mice NO COLITIS log 10 CFU / g feces! 10! 5! 0! + Antibiotics + E. coli + T cells log 10 CFU / g feces 10! 5! 0! COLITIS Increase of ratio Enterobacteriaceae to Bacteroidetes : Induces colitis Julia Frick Gronbach et al. (unpublished) 27
28 Microbiome Analysis : First Results " Pilot sequencing: S reads per sample Colitis n=4 No colitis No colitis n=4 Colitis " Will sequence metagenomic DNA and cdna 28
29 Microbiome Analysis : First Results " Clustering analysis of datasets: Colitis No colitis 29
30 Overview Metagenomics Adipositas Inflammatory bowel disease Waste water treatment
31 Waste Water Metagenomics " Singapore Center for Environmental Life Science Engineering SCELSE ( " Joint NTU NUS Center or Research Excellence, located at NTU Aim: to explore and translate the life of microbial biofilms into environmental solugons " Flagship project: " Analysis of microbial community in the Ulu Pandan water treatment plant 31
32 Technical Replicates " One bucket of sludge five samples from bucket two extracgons & libraries per sample = ten libraries Initial sequencing: 1 mio 454 reads per dataset 32
33 Taxonomic Reproducibility 33
34 Func=onal Reproducibility 34
35 More Detailed Analysis " New Chloroflexi Species GC content Cloud Depth of coverage Assembled data by depth of coverage vs GC content Fangqing Zhao 35
36 More Detailed Analysis MEGAN analysis of Cloud-0: >50% reads are Chloroflexi 36
37 Computa=onal Goals " IdenGfy main players in water treatment microbiome " Characterize previously unknown species " Who is doing what? " Who is cooperagng with whom? " Bio monitoring " UlGmate goal: improve efficiency of water treatment 37
38 Thanks to: " Suparna Mitra, Hans Ruscheweyh & Nico Weber Tübingen " Julia Frick and Bärbel Stecher Tübingen/Munich " Karin Förster- Fromme & Stephan Bischoff Hohenheim " Xie Chao, Daniela Drautz, Peter Liyle, Stephan Schuster, Rohan Williams & Fangqing Zhao Singapore BMBF SCELSE 38
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