Microbiome 101: Sequencing, Nomenclature, and Methodology

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2 Microbiome 101: Sequencing, Nomenclature, and Methodology Ian Carroll, PhD Assistant Professor Division of Gastroenterology and Hepatology UNC Chapel Hill

3 Background

4 Microbiota: an ecological community of commensal, symbiotic and pathogenic microorganisms within a specific region

5 Surface Area of the Gastrointestinal Tract Small Intestine Large Intestine Approx. 270 m 2

6 Epithelial nuclei Microbial Niches Within the Intestinal Tract E. coli 16S rrna probe

7 Important Definitions The Intestinal Microbiota: A complex community of bacteria, archaea, and eukarya that colonize the entire alimentary canal different species. 10 times the number of human cells. The Intestinal Microbiome: The cumulative genomes of the intestinal microbiota. Microbiome contains 100 times the number of genes in the human genome.

8 Intestinal Lumen Competitive exclusion Digestion of food Intestinal physiology Immune system development ORGAN: A part of an organism that is typically selfcontained and has a specific vital function, such as the heart or liver in humans.

9 Sequencing

10 Basis for Molecular Techniques 16S rrna gene Found in all bacteria

11 Sequencing microbial 16S rrna PCR product PCR product

12 clone and sequence V1 V2 V3 V4 V5 V6 V7 V8 V9 V1 V2 V6 Quantitative real-time PCR (qpcr). Sequencing of the 16S rrna gene. High throughput sequencing of the 16S rrna gene.

13 High throughput sequencing of intestinal samples 16S rrna gene OR One 454 Titanium plate 1,000,000 16S rrna reads One Illumina hiseq read 6,000,000,000 16S rrna reads

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15 16S rrna pipeline Diversity Community ecologists typically describe the microbial diversity within their study. This diversity can be assessed: Taxon Within a sample (alpha diversity) Between a collection of samples (beta diversity). Assign high-throughput sequencing reads to taxonomic identities using established databases

16 Average UniFrac distance PC2 PC2 Beta-diversity: Average UniFrac Distances PC1 PC1

17 Visualization of Alpha Diversity Rarefaction Analysis No. of species No. sequences sampled 1000 Carroll. et al. Neurogastroenterol Motil

18 Methods

19 Experimental Design for Analysis of the Fecal Microbiota Frozen Fecal Sample DNA Isolation Sequence Generation Sequence Analysis

20 Different Fecal Microbial DNA Isolation Methods

21 Fecal Microbial DNA Isolation Methods Generate Different Community Diversities METHODS SUBJECTS 1-5 REPLICATES A,B&C Conclusion: Clustering is based on individual and not isolation method Salonen et al J Microbial Methods.

22 Isolation of Fecal Microbial DNA at UNC-CH Current Protocol Qiagen Stool DNA isolation kit Proteinase K SDS Bead Beating Heat 65 Gram Positive Gram Negative Gram Positive DNA Gram Negative DNA Heat 95

23 Methods Continued Proteinase K SDS Bead Beating Heat 65

24 Comparison of Isolation Methods Bacteroides Clostridia Lactobacillus Bifidobacteria E. coli Stool Qiagen Kit 2.85E E E E E+06 CURRENT PROTOCOL 4.78E E E E E+05 Concentrations expressed as number of 16S sequences/µg DNA.

25 Advantages and Disadvantages of Methods Current Protocol Qiagen Stool DNA isolation kit Advantage Disadvantage Advantage Disadvantage 8 hours 3 hours High conc. DNA High diversity of microbes Relatively pure DNA Very pure DNA Low conc. DNA Low diversity of microbes

26 Typical Example of Methods in Literature Community DNA preparation Faecal samples were frozen immediately after they were produced. De-identified samples were stored at -80 C before processing. Ten to twenty grams of each sample was pulverized in liquid nitrogen with a mortar and pestle. An aliquot (approximately 500 mg) of each sample was then suspended, while frozen, in a solution containing 500 μl of extraction buffer (200 mm Tris (ph 8.0), 200 mm NaCl, 20 mm EDTA), 210 μl of 20% SDS, 500 μl of a mixture of phenol:chloroform:isoamyl alcohol (25:24:1, ph 7.9), and 500 μl of a slurry of 0.1-mm diameter zirconia/silica beads (BioSpec Products). Microbial cells were subsequently lysed by mechanical disruption with a bead beater (BioSpec Products) set on high for 2 min at room temperature, followed by extraction with phenol:chloroform:isoamyl alcohol, and precipitation with isopropanol. DNA obtained from three separate aliquots of each faecal sample were pooled ( 200 μg DNA) and used for pyrosequencing (see below). Turnbaugh et al Nature.

27 Experimental Design for Analysis of the Fecal Microbiota Frozen Fecal Sample DNA Isolation Sequence Generation Sequence Analysis

28 Effect of Time at Different Temperatures on Fecal Microbial Diversity Carroll. et al PLOS ONE.

29 Storage and Fecal Microbial Diversity Carroll. et al PLOS ONE.

30 Storage and Fecal Microbial Diversity Carroll. et al PLOS ONE.

31 Storage and Fecal Microbial Diversity

32 Intestinal Niches

33 Diversity Differences Within Intestinal Niches Healthy controls: 24 fecal samples 24 colonic biopsies Ringel. et al Gut Microbes.

34 Compositional Differences Within Intestinal Niches Ringel. et al Gut Microbes.

35 Functional Impact of the Intestinal Microbiota

36 Gnotobiotics gnostos 'known' bios 'life' Normal Microbiota Germ-free Germ-free Mono-associated Dual-associated Consortium of microbes

37 Functional impact of the intestinal microbiota on adiposity GERM-FREE Normal Gut Microbiota Normal Diet Western Diet Normal Diet Backhed. et al PNAS.

38 Functional impact of the intestinal microbiota on adiposity GERM-FREE Normal Diet Ridaura. et al Science.

39 Energy Extraction Hypothesis Lean Obese More calories extracted Increased fat storage Turnbaugh. et al An obesity-associated gut microbiome with increased capacity for energy harvest. Nature.

40 Approach AN T1 <75% IBW AN T2 >85% IBW Healthy Control Germ-free Characterize Adiposity

41 mesenteric fat weight as % of body weight mesenteric fat weight normalized to food consumption Gonadal weight as % of body weight % gonadal weight normalized to food consumption Adiposity results Taxonomy Species-level 4 3 p= p= T1 1 T2 2 RF39 ML615J-28o Cloacibacillus Pyramidobacter Pseudomonas Haemophilus Campylobacter Desulfovibrio Bilophila Sutterella Betaproteobacteriao Rhodospirillaceae Alphaproteobacteriao Sneathia Veillonella Selenomonas Megamonas Dialister Veillonellaceae Bacteroides Peptostreptococcus T1 T2 p= T1 T2 p= T1 T T1 T2

42 Future of Treatment

43 The Intestinal Microbiota and Health Ilya Ilyich Mechnikov Nobel Laureate in Physiology or Medicine Prolongation of Life: Optimistic Studies that aging is caused by toxic bacteria in the gut certain bacteria could improve the intestinal health of the host (Metchnikoff 1908). Based on this theory, he drank sour milk every day. He attributed the longevity of Bulgarian peasants due to their yogurt consumption

44 Therapies Based on the Intestinal Microbiota Yellow Soup: Fermented stool to treat abdominal diseases Li Shizhen

45 Microbiome analyses Acknowledgements for AN study Eating disorders CB LT Mouse behavioral analysis Mouse adiposity biomarkers PKL IC SK JC Mouse behavioral biomarkers AF

46 Acknowledgements Investigators R. Balfour Sartor Eun Young Huh Yehuda Ringel Tamar Ringel-Kulka Jeremy Herzog Elle Glenny Funding Young Investigators Grant for Probiotics Research FGIMD Seed grant NC TraCS CGIBD pilot feasibility award National Institutes of Health NIDDK K01 CGIBD