6.남영도110923

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6.남영도110923

  1. 1. Comparative Analysis of Korean Human Gut Microbio ta by Barcoded Pyrosequencing Young-Do Nam, PhD. Traditional Food Research Team Korea Food Research Institute
  2. 2. 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
  3. 3. Human Microbiome ProjectHuman 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
  4. 4. Roles of gut microbiotaCo-evolution with these great microbial ecosystems serves important functions for the human host by presentingNutrients from dietsResisting the colonization of pathogensstimulating the proliferation of the intestinal epithelium cellsRegulating fat storage of host.In addition, numerous diseases such asType 1 diabetes (T1D)Inflammatory bowel disease (IBD)Gastric or colonic cancersis known to be linked to dysbiosis of microbial communities
  5. 5. Microbial communities in human body VirusAlmost all the surfaces of the human body are occupied by habitat specific microbesThe colon contains 1011-1012 microbial cells per mL with two orders higher genes than human genesContains the three major domains of life: Eukaryota, Archaea, and Bacteria as well as viruses
  6. 6. Cultivation based analysisOnly 20-40% of bacterial species have been cultivated from the human intestine
  7. 7. 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.
  8. 8. Massive sequencing technologyABI 3730Sanger Capillary Based Seq 70,000 bp/run ?
  9. 9. PyrosequencingDNA Beads are generated using Emulsion PCRDNA Beads are placed in wellsNucleotide sequences are decoded by fluorescence
  10. 10. Multiplex Barcoded Pyrosequencing Sample specific barcode sequence
  11. 11. 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
  12. 12. 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
  13. 13. OTU determination 8,600 PhylotypesTotal 303,402 sequencesAverage 8,427 reads per individualAverage 771 species level phylotypes per individual
  14. 14. Rarefaction and coverages Reaching plateau but not saturatedPatterns Reaching plateau but failing to reach a saturation phaseUnseen OTUs still existed in the original samplesGood’s coverage of overall sequence was 90%Majority of bacterial phylotypes is successfully identified
  15. 15. 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
  16. 16. Family level diversity Ruminococaceae BacteroidaceaeThe most dominant family differed between individuals and the proportion of sequences attributable to the families Prevotellaceae and Ruminococcaceae
  17. 17. Taxa distribution 711 species level phylotypes Ave, 107 Ave, 32.6 Ave, 9.2Diversity of specific taxa at the phylum to genus levels is relatively low but extremely high at the species and strain levels
  18. 18. 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
  19. 19. Core Korean gut micobiota Taxonomic rank aNumber ofPhylum Class Family Species OTUBacteroidetes Bacteroidia Bacteroidaceae Bacteroides galacturonicus 2Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides sp. CO55 1Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides sp. CS3 1Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides uniformis 1Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides vulgatus 2Bacteroidetes Bacteroidia Porphyromonadaceae Parabacteroides merdae 1Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium A1-86 1Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium A2-207 2Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium M21/2 3Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium SL6/1/1 1Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium SSC/2 1Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium T1-815 1Firmicutes Clostridia Clostridiales bacterium Clostridiales bacterium 80/4 1Firmicutes Clostridia Clostridiaceae Clostridiaceae bacterium DJF LS13 1Firmicutes Clostridia Clostridiaceae Clostridium bolteae 1Firmicutes Clostridia Clostridiaceae Clostridium orbiscindens 1Firmicutes Clostridia Clostridiaceae Clostridium saccharolyticum 1Firmicutes Clostridia Eubacteriaceae Eubacterium tenue 1Firmicutes Clostridia Eubacteriaceae Eubacterium ventriosum 1Firmicutes Clostridia Ruminococcaceae Faecalibacterium prausnitzii 4Firmicutes Clostridia Ruminococcaceae Faecalibacterium sp. DJF VR20 2Firmicutes Clostridia Ruminococcaceae Ruminococcus gnavus 1Firmicutes Clostridia Ruminococcaceae Ruminococcus sp. CB3 2Firmicutes Clostridia Ruminococcaceae Ruminococcus sp. CJ60 2Firmicutes Clostridia Ruminococcaceae Ruminococcus sp. K-1 1Firmicutes Clostridia Ruminococcaceae Ruminococcus sp. SC103 1Firmicutes Clostridia Ruminococcaceae Subdoligranulum sp. DJF VR33k2 1Firmicutes Unclassified Unclassified Firmicutes bacterium EG20 1Unclassified Unclassified Unclassified Human intestinal bacterium PUE 1Unclassified Unclassified Unclassified Human intestinal firmicute CO35 2Unclassified Unclassified Unclassified Rumen bacterium 8/9293-21 1
  20. 20. Host specificity of gut microbiota The un-weighted UPGMA clustering analysis revealed that each individual have specific microbial members.
  21. 21. Temporal stability of gut microbiota The composition of gut microbiota ordinarily fluctuated through the life.
  22. 22. Community Comparison 100% Uncultured Ruminococcus 80% Faecalibacterium Clostridium 60% Prevotella Bacteroides 40% Bifidobacterium 20% 0% Korea China US JapanEach country member clustered together.Korean takes higher dietary fibers than other country membersKorean have higher abundance of Butyrate producing bacteria
  23. 23. Summary of this studyOverall composition of Korean gut microbiota is similar to that of other countrymembersKorean 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 lifeEeach country member has region specific gut microbiota. And Korean s havehigher level of butyrate producing bacteria rather than other county membersThe difference of gut microbiota seems to come from factors of host genotypeand diet styles
  24. 24. Thank you!

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