Microbiome analysis: from concept to completion. Matthew Stoll MD,PhD,MSCS January 26, 2015

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1 Microbiome analysis: from concept to completion Matthew Stoll MD,PhD,MSCS January 26, 2015

2 We re surrounded by bugs Human body contains 100 trillion microbes Out-number human cells 10:1 Human gut alone: ~ 99% of all of the microbiota 3 pounds of bacteria 50% of fecal volume ~ 1000 different species 3 million different genes (100x human host) Scher, Nat Rev Rheumatol 2011;7:569 De Cruz, Inflamm Bowel Dis 2012;18:372

3 Are they important? Germ-free state results in altered / absent disease expression in several models of autoimmune diseases. IBD Spondyloarthritis with colitis Rheumatoid arthritis Type I Diabetes Multiple sclerosis Wu, Gut Microbes 2012;3:1

4 Microbiota and human inflammatory diseases Inflammatory bowel disease Type I DM Arthritis Allergies / asthma Cancer Celiac disease Clemente, Cell 2012;148:1258

5 Microbiota may impact noninflammatory diseases Obesity 1 Metabolic syndrome 2 Non-alcoholic steatohepatosis 3 Fatty liver Autism 4 1 Turnbaugh, Nature 2006;444: Vrieze, Gastroenterol 2012; 143:913 3 Paolella, World J Gastroenterol 2014;20: Hsiao, Cell 2013;155:1451

6 Getting started Talk with someone who s done it Contact Casey, Elliot Important decisions at outset Habitat (e.g. skin, gut, oral, genitourinary) Controls: may already exist Sample size / power analyses

7 Bacteria composition differs by site HMP Consortium, Nature 2012;486:207

8 Skin has topographically distinct microbiota Heel Elbow Back Findley, Nature 2013;498:367

9 So does the mouth Buccal mucosa Keratinized gingiva Hard palate Throat Tonsils Tongue Dorsum Saliva Segata, Genome Biol 2012;13:R42 Supragingival plaque Subgingival plaque

10 Genitourinary Send subject home with cotton swab

11 Intestinal microbiota: Feces or colonoscopy What do you have access to Bias in selection of patients / controls Affect of washout? Jalanka, Gut 2014;ePub

12 Fecal microbiota Need to overcome the eeew! factor Patient prepares own sample Usually at home Shipment Money helps $25 payment $50 payment Received / Promised 27 / 62 (44%) 128 / 178 (72%)

13 Stool collection Place over toilet

14 Stool collection Scoop

15 Stool collection Add media Tape it up

16 Stool collection Open Therapak box Insert biohazard bag Add cold pack

17 Stool collection Mail in Fed Ex ClinPak Dispose of trash

18 Getting started Stool collected from Three children with ERA One child with psoriasis + joint pain Two adult controls 16S sequencing performed

19 Kingdom ERA ERA Psor. ERA Ctrl Ctrl

20 SpA SpA Psor. SpA Ctrl Ctrl Phylum Verrucomicrobia

21 Family Verrucomicrobiaceae (Akkermansia muciniphila) SpA SpA Psor. SpA Ctrl Ctrl

22 Start of a story 6 yo female with psoriasis and joint pain Subsequently developed abdominal pain Diagnosed with IBD A. muciniphila chews up protective mucin layer in intestines Mucin-deficient mice develop colitis (van der Sluis, Gastroenterology 2006;131:117)

23 Next set of data 16S sequencing performed on 3 pediatric subjects with SpA 4 healthy control subjects

24 Increased bacteroidacaea 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% SpA SpA SpA Ctrl Ctrl Ctrl Ctrl

25 Preliminary data => grant Power calculations were performed using the Dirichlet-Multinomial test a sample size of n = 25 / group would yield statistical power of 0.85 Alternatively, could use prelim data to power to a limited number of organisms using standard approaches (t-test)

26 Next study Feature SpA Controls n Age (years;median,range) 14, , Male:female 13:14 6:7 BMI (median,range) 20.4, , Disease duration 2.4, N/A (years;median,range) Immunosuppresive therapy 20/27 (74%) 0/13

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30 Unsupervised cluster analysis

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32 Table Species SpA Cluster 1 SpA Cluster 2 p-value n 8 19 N/A F. prausnitzii (% of total bacteria, median) Bacteroides (% of total bacteria, median) Akkermansia (fraction 2%) 3.2% 4.4% % 13% < /8 8/

33 Summary of findings Decreased Faecalibacterium prausnitzii Increased in two bacteria identified as being potentially relevant in the preliminary data Bacteroides genus Akkermansia muciniphila

34 Next directions Is dysbiosis an epiphenomenon? Transfer microbiota to arthritis model in gnotobiotic facility (NIH / NIEHS) Does treatment alter the microbiota Collaborative study of newly diagnosed subjects (Friends of CARRA) Immunologic affects of microbiota Are there functional alterations? Metagenomics Metabolomics (Steve Barnes)

35 Summary / conclusions Meet with stats in advance, also with researchers studying same microbiota mstoll@peds.uab.edu Incentives help! With funding agencies, be prepared to answer the so what question, what comes next after you show differences