6.남영도110923

Comparative Analysis of Korean Human Gut Microbiota by Barcoded Pyrosequenc-

ing

Young-Do Nam, PhD.

Traditional Food Research Team

Korea Food Research Institute

Human genome project

(1.8 Million Bases)

106 107 108105 109 1010DNA Base Pairs

Russell Dolittle, Nature .419, p. 494 (2002)

Human genome contains around 30,000 genes rather than the estimated 100,000 protein coding genes

Genome size :3.3 Billion BasesBut only contains 30,000 genes

Microbes

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 be-tween human health and these gut microbiota

Roles of gut microbiota

Co-evolution with these great microbial ecosystems serves im-portant 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 cancers

is known to be linked to dysbiosis of microbial communities

Microbial communities in human body

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 genesContains the three major domains of life: Eukaryota, Archaea, and Bacteria as well as viruses

Virus

Cultivation based analysis

Only 20-40% of bacterial species have been cultivated from the human intestine

History of molecular methods

19771953

Watson&Crick 의 DNA double helix

1875

Cohn,Pasteur,Koch,

Metchnikoff

Jacob&Monod

lac operon

1960

Sanger de-termine the sequence of all 5,375 nu-cleotides of

bacteriophage phi-X174, the first complete genome of an

organism.

Carl Woese uses ribosomal

RNA analysis to recognize a third form of life, the

Archaea

Kary Mullis uses a heat

stable enzyme from Thermus aquaticus to

establish polymerase

chain reaction technology.

1986

Craig Venter at TIGR eluci-date the first

complete genome se-quence of a

microorganism: Haemophilus

influenza.

19951993

Norman Pace uses rRNA as a tool for mi-crobial ecol-

ogy.

1989

Ed Delong developed a powerful

method; FISH

G. Muyzer DGGE :the

most useful in current mi-

crobial ecol-ogy

Mark Shena:Microarrays prepared by

high-speed ro-botic printing

of complemen-tary DNAs on

glass

C. Venter se-quenced the

human genome using the whole genome shot-gun technique

1998 2002

James Tiedje report the first environmental

microarray

ABI 3730

Sanger Capillary Based Seq

70,000 bp/run

Massive sequencing technol-ogy

?

Pyrosequencing

DNA Beads are generated using Emulsion PCR

DNA Beads are placed in wells

Nucleotide sequences are decoded by fluorescence

Multiplex Barcoded Pyrosequenc-ing

Sample specific barcode sequence

Data analysis pipelineBarcoded

pyrosequencing data

Trimming quality filtering

Community Comparison

(UniFrac, UPGMA)

Barcode sorting

alignment

OTU determination

Merge fileDistance

matrix

Classification,Diversity estimation

Community composition (Diversity)

DNA extraction

Sample collec-tion

Amplification With barcoded

primer

Pyro-sequencing

Wet-lab analysis

Data analysis pipelineBarcoded

pyrosequencing data

Trimming quality filtering

Community Comparison

(UniFrac, UPGMA)

Barcode sorting

alignment

OTU determination

Merge fileDistance

matrix

Classification,Diversity estimation

Community composition (Diversity)

DNA extraction

Sample collec-tion

Amplification With barcoded

primer

Pyro-sequencing

Dry-lab analysis

OTU determination

Spec

ies

leve

l

Gen

us le

vel

Fam

ily le

vel

8,600 Phylotypes

Total 303,402 sequences

Average 8,427 reads per individual

Average 771 species level phylotypes per individual

Rarefaction and coverages

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

Reaching plateau but not saturated

Phylum level diversi-ties

The phylum level diversity of Korean gut microbiota is similar to the other human populations

Eckburg et al. Science (2005)

From Ley et al. Cell (2006)

Bacteroidetes

Firmicutes

Family level diversity

The most dominant family differed between individuals and the proportion of sequences attributable to the families Prevotellaceae and Ruminococcaceae

Bacteroidaceae

Ruminococaceae

Taxa distribution

Diversity of specific taxa at the phylum to genus levels is relatively low but extremely high at the species and strain levels

Ave, 9.2

Ave, 32.6

Ave, 107

711 species level phylotypes

ID Core gut microbiota1 Bacteroides2 Parabacteroides3 Prevotella 4 Uncultured butyrate-producing bacterial group5 Uncultured human intestinal Firmicutes group6 Clostridium 7 Eubacterium8 Faecalibacterium9 Lachnospira

10 Oscillibacter11 Roseburia12 Ruminococcus13 Subdoligranulum14 Fusobacterium

Core Korean gut micobiota

Core Korean gut micobiotaTaxonomic rank aNumber of

OTUPhylum Class Family Species

Bacteroidetes 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

Host specificity of gut microbiota

The un-weighted UPGMA cluster-ing analysis revealed that each in-dividual have specific microbial members.

The composition of gut microbiota ordinarily fluctu-ated through the life.

Temporal stability of gut micro-biota

Community Comparison

Each country member clustered together.

Korean takes higher dietary fibers than other country members

Korean have higher abundance of Butyrate producing bacteria

Korea China US Japan0%

20%

40%

60%

80%

100%Uncultured

Ruminococcus

Faecalibacterium

Clostridium

Prevotella

Bacteroides

Bifidobacterium

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 diver-sity at species level.

The member of korean gut microbiota stably maintained but the abundance of each community member seems to continuously fluctu-ate through the life

Eeach country member has region specific gut microbiota. And Ko-rean 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

Thank you!