Microbiome 101: Sequencing, Nomenclature, and Methodology Ian Carroll, PhD Assistant Professor Division of Gastroenterology and Hepatology UNC Chapel Hill
Background
Microbiota: an ecological community of commensal, symbiotic and pathogenic microorganisms within a specific region
Surface Area of the Gastrointestinal Tract Small Intestine Large Intestine Approx. 270 m 2
Epithelial nuclei Microbial Niches Within the Intestinal Tract E. coli 16S rrna probe
Important Definitions The Intestinal Microbiota: A complex community of bacteria, archaea, and eukarya that colonize the entire alimentary canal. 300-1000 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.
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.
Sequencing
Basis for Molecular Techniques 16S rrna gene Found in all bacteria
Sequencing microbial 16S rrna PCR product PCR product
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.
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
http://qiime.sourceforge.net/
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
Average UniFrac distance PC2 PC2 Beta-diversity: Average UniFrac Distances PC1 PC1
Visualization of Alpha Diversity Rarefaction Analysis No. of species 30 20 10 10 100 No. sequences sampled 1000 Carroll. et al. Neurogastroenterol Motil. 2012.
Methods
Experimental Design for Analysis of the Fecal Microbiota Frozen Fecal Sample DNA Isolation Sequence Generation Sequence Analysis
Different Fecal Microbial DNA Isolation Methods
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. 2010 J Microbial Methods.
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
Methods Continued Proteinase K SDS Bead Beating Heat 65
Comparison of Isolation Methods Bacteroides Clostridia Lactobacillus Bifidobacteria E. coli Stool Qiagen Kit 2.85E+08 1.90E+08 1.29E+04 5.99E+06 2.33E+06 CURRENT PROTOCOL 4.78E+07 3.51E+08 2.13E+06 6.99E+07 5.63E+05 Concentrations expressed as number of 16S sequences/µg DNA.
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
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. 2008 Nature.
Experimental Design for Analysis of the Fecal Microbiota Frozen Fecal Sample DNA Isolation Sequence Generation Sequence Analysis
Effect of Time at Different Temperatures on Fecal Microbial Diversity Carroll. et al. 2012 PLOS ONE.
Storage and Fecal Microbial Diversity Carroll. et al. 2012 PLOS ONE.
Storage and Fecal Microbial Diversity Carroll. et al. 2012 PLOS ONE.
Storage and Fecal Microbial Diversity
Intestinal Niches
Diversity Differences Within Intestinal Niches Healthy controls: 24 fecal samples 24 colonic biopsies Ringel. et al. 2015 Gut Microbes.
Compositional Differences Within Intestinal Niches Ringel. et al. 2015 Gut Microbes.
Functional Impact of the Intestinal Microbiota
Gnotobiotics gnostos 'known' bios 'life' Normal Microbiota Germ-free Germ-free Mono-associated Dual-associated Consortium of microbes
Functional impact of the intestinal microbiota on adiposity GERM-FREE Normal Gut Microbiota Normal Diet Western Diet Normal Diet Backhed. et al. 2006 PNAS.
Functional impact of the intestinal microbiota on adiposity GERM-FREE Normal Diet Ridaura. et al. 2013 Science.
Energy Extraction Hypothesis Lean Obese More calories extracted Increased fat storage Turnbaugh. et al. 2006 An obesity-associated gut microbiome with increased capacity for energy harvest. Nature.
Approach AN T1 <75% IBW AN T2 >85% IBW Healthy Control Germ-free Characterize Adiposity
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=0.06 0.025 0.020 p=0.03 1 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0 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 2 1 0 20 15 10 5 T1 T2 p=0.02 0.015 0.010 0.005 0.000 0.15 0.10 0.05 T1 T2 p=0.02 0 T1 T2 0.00 T1 T2
Future of Treatment
The Intestinal Microbiota and Health Ilya Ilyich Mechnikov - 1908 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
Therapies Based on the Intestinal Microbiota Yellow Soup: Fermented stool to treat abdominal diseases Li Shizhen 1518-1593
Microbiome analyses Acknowledgements for AN study Eating disorders CB LT Mouse behavioral analysis Mouse adiposity biomarkers PKL IC SK JC Mouse behavioral biomarkers AF
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