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

6. Cultivation based analysis
Only 20-40% of bacterial species have been cultivated from
the human intestine

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.

8. Massive sequencing technology
ABI 3730
Sanger Capillary Based Seq
70,000 bp/run ?

9. Pyrosequencing
DNA Beads are generated using Emulsion PCR
DNA Beads are placed in wells
Nucleotide sequences are decoded by fluorescence

10. Multiplex Barcoded Pyrosequencing
Sample specific barcode sequence

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

19. Core Korean gut micobiota
Taxonomic rank aNumber of
Phylum Class Family Species OTU
Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides galacturonicus 2
Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides sp. CO55 1
Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides sp. CS3 1
Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides uniformis 1
Bacteroidetes Bacteroidia Bacteroidaceae Bacteroides vulgatus 2
Bacteroidetes Bacteroidia Porphyromonadaceae Parabacteroides merdae 1
Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium A1-86 1
Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium A2-207 2
Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium M21/2 3
Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium SL6/1/1 1
Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium SSC/2 1
Firmicutes Clostridia Butyrate-producing bacterium Butyrate-producing bacterium T1-815 1
Firmicutes Clostridia Clostridiales bacterium Clostridiales bacterium 80/4 1
Firmicutes Clostridia Clostridiaceae Clostridiaceae bacterium DJF LS13 1
Firmicutes Clostridia Clostridiaceae Clostridium bolteae 1
Firmicutes Clostridia Clostridiaceae Clostridium orbiscindens 1
Firmicutes Clostridia Clostridiaceae Clostridium saccharolyticum 1
Firmicutes Clostridia Eubacteriaceae Eubacterium tenue 1
Firmicutes Clostridia Eubacteriaceae Eubacterium ventriosum 1
Firmicutes Clostridia Ruminococcaceae Faecalibacterium prausnitzii 4
Firmicutes Clostridia Ruminococcaceae Faecalibacterium sp. DJF VR20 2
Firmicutes Clostridia Ruminococcaceae Ruminococcus gnavus 1
Firmicutes Clostridia Ruminococcaceae Ruminococcus sp. CB3 2
Firmicutes Clostridia Ruminococcaceae Ruminococcus sp. CJ60 2
Firmicutes Clostridia Ruminococcaceae Ruminococcus sp. K-1 1
Firmicutes Clostridia Ruminococcaceae Ruminococcus sp. SC103 1
Firmicutes Clostridia Ruminococcaceae Subdoligranulum sp. DJF VR33k2 1
Firmicutes Unclassified Unclassified Firmicutes bacterium EG20 1
Unclassified Unclassified Unclassified Human intestinal bacterium PUE 1
Unclassified Unclassified Unclassified Human intestinal firmicute CO35 2
Unclassified Unclassified Unclassified Rumen bacterium 8/9293-21 1

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

24. Thank you!