Comparative Analysis of Korean Human Gut Microbio ta by Barcoded Pyrosequencing Young-Do Nam, PhD. Traditional Food Research Team Korea Food Research Institute
Human genome project 105 106 107 108 109 1010 DNA Base Pairs Genome size :3.3 Billion Bases But only contains 30,000 genesMicrobes (1.8 Million Bases) Russell Dolittle, Nature .419, p. 494 (2002)Human genome contains around 30,000 genes rather than the estimated 100,000 protein coding genes
Human Microbiome ProjectHuman Microbiome Project (HMP) was initiated in US, Europe, Japan, China and many other countries.To characterize “microbiome” and examine the relatedness between humanhealth and these gut microbiota
Roles of gut microbiotaCo-evolution with these great microbial ecosystems serves important functions for the human host by presentingNutrients from dietsResisting the colonization of pathogensstimulating the proliferation of the intestinal epithelium cellsRegulating fat storage of host.In addition, numerous diseases such asType 1 diabetes (T1D)Inflammatory bowel disease (IBD)Gastric or colonic cancersis known to be linked to dysbiosis of microbial communities
Microbial communities in human body VirusAlmost all the surfaces of the human body are occupied by habitat specific microbesThe colon contains 1011-1012 microbial cells per mL with two orders higher genes than human genesContains the three major domains of life: Eukaryota, Archaea, and Bacteria as well as viruses
Cultivation based analysisOnly 20-40% of bacterial species have been cultivated from the human intestine
History of molecular methods Carl Woese G. Muyzer Norman Ed Delong uses ribosomal DGGE :the Cohn, Pace uses James Tiedje RNA analysis to developed most useful in Pasteur, recognize a third rRNA as a a powerful current report the first Koch, tool for method; environmental form of life, the microbialMetchnikoff microbial FISH microarray Archaea ecology ecology. 1875 1953 1960 1977 1986 1989 1993 1995 1998 2002 Watson&Crick의 Jacob&Monod Sanger Kary Mullis Craig Venter Mark Shena: C. Venter DNA double helix determine the uses a heat at TIGR Microarrays sequenced the lac operon sequence of stable enzyme elucidate the prepared by human genome all 5,375 from Thermus first complete high-speed using the whole nucleotides of aquaticus to genome robotic printing genome bacteriophage establish sequence of a of shotgun phi-X174, the polymerase microorganism: complementary technique first complete chain reaction Haemophilus DNAs on glass genome of an technology. influenza. organism.
PyrosequencingDNA Beads are generated using Emulsion PCRDNA Beads are placed in wellsNucleotide sequences are decoded by fluorescence
Multiplex Barcoded Pyrosequencing Sample specific barcode sequence
Data analysis pipeline Barcoded pyrosequencing Sample collection data Trimming quality filtering DNA extraction Barcode sorting alignment OTU Amplification determination With barcoded Merge file Classification, primer Distance Diversity matrix estimation Community Community Pyro-sequencing Comparison composition (UniFrac, UPGMA) (Diversity)Wet-lab analysis
Data analysis pipeline Barcoded pyrosequencingSample collection data Trimming quality filtering DNA extraction Barcode sorting alignment OTU Amplification determination With barcoded Merge file Classification, primer Distance Diversity matrix estimation Community CommunityPyro-sequencing Comparison composition (UniFrac, UPGMA) (Diversity) Dry-lab analysis
OTU determination 8,600 PhylotypesTotal 303,402 sequencesAverage 8,427 reads per individualAverage 771 species level phylotypes per individual
Rarefaction and coverages Reaching plateau but not saturatedPatterns Reaching plateau but failing to reach a saturation phaseUnseen OTUs still existed in the original samplesGood’s coverage of overall sequence was 90%Majority of bacterial phylotypes is successfully identified
Phylum level diversities Firmicutes Bacteroidetes Eckburg et al. Science (2005) From Ley et al. Cell (2006)The phylum level diversity of Korean gut microbiota is similar to the other human populations
Family level diversity Ruminococaceae BacteroidaceaeThe most dominant family differed between individuals and the proportion of sequences attributable to the families Prevotellaceae and Ruminococcaceae
Taxa distribution 711 species level phylotypes Ave, 107 Ave, 32.6 Ave, 9.2Diversity of specific taxa at the phylum to genus levels is relatively low but extremely high at the species and strain levels
Core Korean gut micobiota ID Core gut microbiota 1 Bacteroides 2 Parabacteroides 3 Prevotella 4 Uncultured butyrate-producing bacterial group 5 Uncultured human intestinal Firmicutes group 6 Clostridium 7 Eubacterium 8 Faecalibacterium 9 Lachnospira 10 Oscillibacter 11 Roseburia 12 Ruminococcus 13 Subdoligranulum 14 Fusobacterium
Host specificity of gut microbiota The un-weighted UPGMA clustering analysis revealed that each individual have specific microbial members.
Temporal stability of gut microbiota The composition of gut microbiota ordinarily fluctuated through the life.
Community Comparison 100% Uncultured Ruminococcus 80% Faecalibacterium Clostridium 60% Prevotella Bacteroides 40% Bifidobacterium 20% 0% Korea China US JapanEach country member clustered together.Korean takes higher dietary fibers than other country membersKorean have higher abundance of Butyrate producing bacteria
Summary of this studyOverall composition of Korean gut microbiota is similar to that of other countrymembersKorean individuals contain diverse and host specific microbial diversity atspecies level.The member of korean gut microbiota stably maintained but the abundance ofeach community member seems to continuously fluctuate through the lifeEeach country member has region specific gut microbiota. And Korean s havehigher level of butyrate producing bacteria rather than other county membersThe difference of gut microbiota seems to come from factors of host genotypeand diet styles