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. Human genome project
105 106 107 108 109 1010 DNA Base Pairs
Genome size :3.3 Billion Bases
But only contains 30,000 genes
Microbes
(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. 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 human
health and these gut microbiota
4. Roles of gut microbiota
Co-evolution with these great microbial ecosystems serves important fu
nctions 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 cancers
is known to be linked to dysbiosis of microbial communities
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
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 microbial
Metchnikoff 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.
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. 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)
Dry-lab analysis
13. OTU determination
8,600 Phylotypes
Total 303,402 sequences
Average 8,427 reads per individual
Average 771 species level phylotypes per individual
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. 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. 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. 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. 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
20. Host specificity of gut microbiota
The un-weighted UPGMA clustering
analysis revealed that each individual have
specific microbial members.
21. Temporal stability of gut microbiota
The composition of gut microbiota ordinarily fluctuated
through the life.
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. Summary of this study
Overall composition of Korean gut microbiota is similar to that of other country
members
Korean individuals contain diverse and host specific microbial diversity at
species level.
The member of korean gut microbiota stably maintained but the abundance of
each community member seems to continuously fluctuate through the life
Eeach country member has region specific gut microbiota. And Korean s have
higher level of butyrate producing bacteria rather than other county members
The difference of gut microbiota seems to come from factors of host genotype
and diet styles
