presentation:dr. liu, wen-tso · ast seminar in thailand, 11-12 mar 08 molecular biological tools...

1

Division of Environmental Science & Engineering

HOPE(s) for QantitativeEnvironmental

Microbiological Studies

Wen-Tso Liu

AST seminar in Thailand, 11-12 Mar 08

Molecular Biological Tools (MBT)

Nucleic Acid based Whole cell based

- DNA/RNA extraction- DNA/RNA quantification

- Quantitation• q-PCR• q-rt PCR

- Fingerprinting• DGGE• T-RFLP• LH-PCR• microarray

- Sequencing• Clone library• Metagenomics

- Cell fixation

- FISH• NA-based• immuno-assay

- MAR- SIP

- Detection System• Optical microscope• RAMAN scope• Flow Cytometry

sample

large volume? yes Conc.

no purification

Intact cell detection

nucleic acid/ protein/metab

olite based detection

cell lysis

Sample collection & concentration

Sample preparation/ secondary concentration

Detection/ analysis

Challenges- types of targets- QA/QC

- Reproducibility- Reliability

- Ease of use- Sensitivity/specificity

- lowest amount/conc.- 1-bp

- Quantitation- Resolution (phylogeny)- Time- Cost- Safety- lab use v.s. on-site tools

- clone library v.s. fingerprinting methods

Other “Omics”

2


BB

AA

CC

Environmental sampleEnvironmental sample

(1) DNA extraction

Mixture of microbial DNAMixture of microbial DNA

(3) Restriction enzyme digestion

5’

3’

5’

5’

3’

3’

5’

3’

3’

5’

3’

5’

1200 bp 300 bp

1100 bp 400 bp

600 bp 800 bp

100 bp

Cut at specific siteCut at specific site

C

B

A600

1100

1200

Fluo

resc

ent i

nten

sity

(RFU

)

(4) Separation fragments by electrophoresis

Fragments on gel after Fragments on gel after electrophoresiselectrophoresis

(5) Detection of fluorescent fragments

Typical Typical electropherogramselectropherograms of of TT--RFLP fingerprintRFLP fingerprint

1200 bp1100 bp800 bp

600 bp

400 bp300 bp

100 bp

5’

5’

3’

3’

5’

5’

3’

3’

5’

5’

3’

3’

FluorescentFluorescent PCR products PCR products of a specific gene of a specific gene

(2) PCR with a fluorescently labeledforward primer

Image analysisImage analysisCluster analysisCluster analysisPrincipalPrincipal--component analysiscomponent analysisWebWeb--based database analysisbased database analysis

http://rdp.cme.eduhttp://rdp.cme.eduhttp://hermes.campus.uidaho.eduhttp://hermes.campus.uidaho.eduhttp://trflp.limnology.wisc.eduhttp://trflp.limnology.wisc.eduhttp://www.oardc.ohiohttp://www.oardc.ohio--state.edu/trflpfragsortstate.edu/trflpfragsort//

(6) Data analysis

Outline of Outline of TT--RFLPRFLP

(Liu et al., 1997)(Liu et al., 1997)


Novel quantitative approach

• Easy

• Fast

• Multiplexing

Constraints of current molecular quantitative techniques

The difficulty to conduct multiplexing analysis is the most severe bottleneck for launching large-scale investigation of microbial abundances.

3


Hierarchical Oligonucleotide Primer Extension (HOPE) (Wu and Liu, NAR, 2007)

Acknowledgement- Wu Jer-Horng- Hong Pei-Ying- NUS funding

Outline- concept- hierarchical primer design- specificity- sensitivity- multiplexing


Sequence specific fluorescence tag labeling by incorporating dye-terminators to 3’end of primers

Clean up dye-terminator-tagged products

PCR amplicons (e.g., 16S rRNA gene)

Purification

Dye-Terminators(Fluorophore-labeled ddNTPs)

ddATPddTTPddCTPddGTP

CT

G

APrimer: 5’ --- 3’

DNA PolymerasePol

P1P2P3

Measure labeled primers by gel-based DNA sequencer

ACTG

PolC

3’CG TA

TA

Pol

3’ GC CPol Pol

5’ Target a

5’ Target c

A

3’ CG GCPol Pol5’ Target b

GT C

Pol

Inte

nsit

y

Size

Internal standardsP1* P2*

P3*

3’ CG AT

3’ CG GC

3’ C

5’ Target b

5’ Target a

5’ Target c

30~4

5 m

in30

-70

min

40

min

Tota

l tim

e re

quire

d: 2

~3 h

r

10

min

CG

4


A

C

TG

GC

C3’ G 5’ Target



CT

G

AOligonucleotide

ProbeDNA

Polymerase

Hierarchical Oligonucleotide Primer Extension- mini-sequencing or single base extension

C 1st cycleC 2nd cycle

C n cycle

Inte

nsity

Size

Internal standards

Capillary Electrophoresis-Laser Induced Fluorescence (CE-LIF) Detection


P1

P2

P3

Hierarchical Oligonucleotide Primer Extension(HOPE)



CT

G

A

OligonucleotideProbe

DNA Polymerase

A

C

TG

GC

T A

AG T

C

G

C T

C

C

3’ G A

3’ C

5’ Target a

5’ Target c

3’ G C 5’ Target b

G

polyTs

Inte

nsity

Size

Internal standardsP1

P2

P3

Capillary Electrophoresis-Laser Induced Fluorescence (CE-LIF) Detection

5


Principle of HOPE

C, final concentration of the extended primer, Co, the initial concentration of target templateE, the extension efficiency, a constantn, the number of thermal cyclesCp2 = Co, p2×E2×n

Cp3 = Co, p3×E3×n

E1, E2, E3, constant, but E1 ≠ E2 ≠ E3

E2 / E1 = a E3 / E1 = b

Cp1 = Co, p1×E1×n

Cp1=aCp2=bCp3

Calibrated


1

0.5

1.5

1

0.5

1.5

P1 P2 P3Template

Hierarchical probes

Ideal Cp1=Cp2=Cp3

ActualCp1=Cp2=Cp3

Factors to synchronize

Cp1=aCp2=bCp3

Calibrated

6


Escherichia coli

Bacteroides fragilis

Bacteroides vulgatus

Bacteroides distasonis

Bifidobacterium

Collinsella

Eubacterium

Low GC

EUB338(79.5% for C*, 9.9% for T*)

EUB338Ia (91.6% for T*)

BAC303 (C*)

Bacteroides thetaiotaomicron

BTH584(C*)

BTH274(C*)

(T*)

Primer Design: Fecal Bacteroides as a model system

o obligate anaerobes o abundant in the faeces of warm-

blooded animalso predict recent fecal contaminationo distinguish fecal pollution sources

from human and livestock.

Indicator for fecal pollution


0

10000

20000

30000

40000

50000

60000

0 10 20 30 40 50 60 70 80 90

EUB

338I

a

BAC

303_

5aB

TH27

4_15

a

BTH

584_

16a

Size (nt)

Fluo

resc

ence

inte

nsity

(rfu

)

Probe name Specificity Binding length Poly dA length Total length

EUB338Ia Bacteria domain 17 0 17

BAC303 Bacteroide-Prevotella group 17 5 22

BTH274 B. thetaiotaomicron

B. fragilis 17 15 32

BTH584 B. thetaiotaomicron 20 16 36

7


Probe-to-target molar ratio100 1000 10000

Con

vers

ion

fact

or (v

.s E

UB

338I

a)

0

2

4

6

8BAC303-5aBTH274-15a BTH584-16a

20000

Conversion factor = constant?

(Fixed probe concentration but varied template concentrations)


Specificity- Target vs. non-target

8


Length

BTH584 Species 5-CAACTGACTTAACTGTCCAC C-3B. thetaiotaomicron 3-GTTGACTGAATTGACAGGTG G-5B. fragilis 3-===========G=T====== G-5B. vulgatus 3-============TGT====A G-5B. distasonis 3-=GC=========TT=C==C= G-5

BTH274Species 5-CCCCTATCCATCGAAGG T-3B. thetaiotaomicron 3-GGGGATAGGTAGCTTCC A-5B. fragilis 3-================= G-5B. vulgatus 3-================T G-5B. distasonis 3-=============AG== A-5

EUB338Species 5-GCTGCCTCCCGTAGGAGT T-3B. thetaiotaomicron 3-CGACGGAGGGCATCCTCA A-5B. fragilis 3-================== A-5B. vulgatus 3-================== A-5B. distasonis 3-================== A-5

BAC303Species 5-CCAATGTGGGGGACCTT C-3B. thetaiotaomicron 3-GGTTACACCCCCTGGAA G-5B. fragilis 3-================= G-5B. vulgatus 3-================= G-5B. distasonis 3-================= G-5

18

0

119

Primer

A tail

Allele

Total

17

5

123

Primer

A tail

Allele

Total

17

15

133

Primer

A tail

Allele

Total

20

16

137

Primer

A tail

Allele

Total

0

10000

20000

30000

40000

50000

60000

0 10 20 30 40 50 60 70 80 90

EUB

BAC

BTH

274

BTH

584

Size (nt)

B. thetaiotamicronEub338 (T) BAC303 (C)BTH274 (T)BTH584 (C)

0

2500

5000

7500

10000

12500

15000

EUB

BA

CB

TH27

4

B. fraglisEub338 (T) BAC303 (C)BTH274 (C)BTH584

B. vulgatusEub338 (T)BAC303 (C)BTH274BTH584

0

2500

5000

7500

10000

12500 EUB

BA

C

0 10 20 30 40 50 60 70 80 90

05000

10000150002000025000300003500040000

0 10 20 30 40 50 60 70 80 90

EUB

BA

C

B. distasonisEub338 (T)BAC303 (C)BTH274BTH584

BTH584


MM2 Allele Size ddNTP MM2 Allele Size3 ddNTP3 MM Allele Size ddNTP MM Allele Size ddNTP

1 B thetaiotaomicron BCRC10624 0 A 17.6 T 0 G 19.9 C 0 A 27.0 T 0 G 31.6 C

2 B. fragilis BCRC10619 0 A 17.5 T 0 G 20.0 C 0 G 27.3 C 2 ND ND

3 B. distasonis JCM5825 0 A 17.7 T 0 G 20.2 C 2 ND ND 6 ND ND

4 B. vulgatus BCRC12903 0 A 17.5 T 0 G 19.9 C 7 ND ND 4 ND ND

5 R. albus DSMZ20455 0 G 16.8 C 6 ND ND 7 ND ND 8 ND ND

6 C. aerofaciens JCM10188 0 G 16.9 C 6 ND ND 7 ND ND 10 ND ND

7 L. acidophilus DSMZ20079 0 A 17.5 T 5 ND ND 7 ND ND 7 ND ND

8 B. adolescentis BCRC14606 0 G 16.9 C 6 ND ND 5 ND ND 9 ND ND

9 P. productus DSM2950 0 G 16.9 C 6 ND ND 8 ND ND 9 ND ND

10 C. leptum BCRC14522 0 G 16.9 C 6 ND ND 7 ND ND 10 ND ND

11 R. bromii ATCC27255 0 G 17.0 C 6 ND ND 7 ND ND 8 ND ND

12 B. longum BCRC11847 0 G 17.0 C 6 ND ND 5 ND ND 9 ND ND

13 E. faecium BCRC10067 0 A 17.5 T 5 ND ND 6 ND ND 7 ND ND

14 C. clostridiiforme BCRC14545 0 A 17.5 T 6 ND ND 6 ND ND 8 ND ND

15 B. longum DSMZ20088 0 G 17.1 C 6 ND ND 5 ND ND 9 ND ND

16 R. obeum ATCC29174 0 G 17.0 C 6 ND ND 7 ND ND 6 ND ND

17 E. biforme DSMZ3989 0 A 17.5 T 6 ND ND 5 ND ND 9 ND ND

18 F. prausnitzii ATCC27768 0 G 17.0 C 5 ND ND 6 ND ND 6 ND ND

19 R. callidus ATCC27760 0 G 16.8 C 5 ND ND 8 ND ND 7 ND ND

20 E. coli NCIMB10083 0 G 16.4 C 6 ND ND 8 ND ND 5 ND ND

17.0 C/T 20.0 C 27.2 C/T 32.0 C

0.30 0.14 0.21 ---

# Species Source1 Eub338 (18nt) BAC303-5a(22nt) BTH274-15a(32nt) BTH584-16a(36nt)

Standard deviation:

Averaged size (nt) and Terminators:

Table 1 Muliplexing single terminator extension analysis of twenty fecal bacterial strains with one set ofhierarchical oligonucleotide probes.

9


Multiplexing

- 4 Plexing- 6 Plexing- 7 Plexing- Multiple tube approach


Multiplexing – theoreticaln (length type) x m (fluorescence type)

ATGCGGGCGCTTGCATGCCGTCCATGCGGGAGTGCGCGCG

ATGCGGGCGCGCCTTGCATGCCGTTATCCATGCGGGTGGAGTGCGCGCGAAT

ATGCGGGCGATCTTTTGCATGCCGGGCGTCCATGCGGGAATTAGTGCGCGCGCCAT

ATGC

ATGC

ATGC

11 14 17

10


Escherichia coli


Bacteroides vulgatus

Bacteroides distasonis

Bifidobacterium

Collinsella

Eubacterium

Low GC

EUB338(79.5% for C*, 9.9% for T*)

EUB338Ia (91.6% for T*)

BAC303 (C*)

Bacteroides thetaiotaomicron

BTH584(C*)

BTH274(C*)

(T*)

Primer Design: Fecal Bacteroides as a model system

o obligate anaerobes o abundant in the faeces of warm-

blooded animalso predict recent fecal contaminationo distinguish fecal pollution sources

from human and livestock.

Indicator for fecal pollution


Model microbial communities.

15.3 ± 1.221.817.4 ± 0.421.853.1 ± 2.946.2AC2 + M. barkeri (12.8 fmol/µl)

3.4 ± 1.05.22.8 ± 0.35.29.7 ± 0.811.1AC2 + E. coli(15.8 fmol/µl)

14.9 ± 1.021.514.4 ± 0.921.514.8 ± 1.021.53AC3

18.1 ± 1.421.817.6 ± 0.721.849.2 ± 3.646.22AC2

18.5 ± 1.222.118.4 ± 0.722.177.0 ± 3.972.71AC1

ObservedTheoreticalObservedTheoreticalObservedTheoretical

BTH584-16a(for B. thetaiotaomicron)

BTH274-15a(for B. thetaiotaomicron)

BAC303-5a(for Bacteroides)

% of the probes for specific groups with respect to probe EUB338Ia for domain Bacteria (mean ± sd, n=6)

Artificial communities

1AC1 contained 16S rRNA gene amplicons of B. thetaiotaomicron (2.2 fmol/µl), B. distasonis (2.5 fmol/µl), B. vulgatus (2.5 fmol/µl) and L. acidophilus (2.7 fmol/µl).

2AC2 contained 16S rRNA gene amplicons of B. thetaiotaomicron (2.2 fmol/µl), B. distasonis (2.5 fmol/µl), L. acidophilus (2.7 fmol/µl) and E. faecium (2.7 fmol/µl).

3AC3 contained 16S rRNA gene amplicons of B. thetaiotaomicron (2.2 fmol/µl), L. acidophilus (2.7 fmol/µl) and E. faecium (2.7 fmol/µl), P. productus (2.7 fmol/µl).

11


●B

TT12

50(T

*)

Bacteroides tectus (tRF 496)Bacteroides pyogenes (tRF 496)Bacteroides suis (tRF 496)Bacteroides denticanum (tRF 494)

Bacteroides nordii (tRF 161)Bacteroides salyersiae (tRF 59)Bacteroides thataiotaomicron (tRF 167)

Bacteroides finegoldii (tRF 495)Bacteroides finegoldii (tRF 495)Bacteroides ovatus (tRF 495)

Bacteroides fragilis (tRF 165)Bacteroides caccae (tRF 166)

Bacteroides acidifaciens (tRF 495)Bacteroides acidifaciens (tRF 167) Bacteroides intestinalis (tRF 489)Bacteroides intestinalis (tRF 489)

Bacteroides helcogenesBacteroides uniformis (tRF 165/493)Bacteroides gallinarum (tRF 165)Bacteroides gallinarum (tRF 165)Bacteroides stercoris (tRF 165)Bacteroides eggerthii (tRF 165)

Bacteroides eggerthii (tRF 165)Bacteroides vulgatus (tRF 165)

Bacteroides vulgatus (tRF 165)Bacteroides dorei (tRF 161)Bacteroides dorei (tRF 161)

Bacteroides massiliensis (tRF 494)Bacteroides massiliensis (tRF 494)Bacteroides coprocola (tRF 494)

Bacteroides plebeius (tRF 494)

Bacteroides barnesiae (tRF 107)

Parabacteroides distasonis (tRF 141)Parabacteroides merdae (tRF 165)Parabacteroides merdae (tRF 165)Parabacteroides johnsonii (tRF 165)

Bacteroides merdae (tRF 165)Parabacteroides goldsteinii

Bacteroides splanchnicus (tRF 489)0.10

○●

EUB

338I

a(T*

)

○●

BA

C30

3(C

*)

●■

BFR

G60

2(C

*)

○●■

BTH

274(

T*)

■BUFM1018(T*)

■BFG1024(C*)

○BTH584(C*)

■BADF1037(G*)

■BITT141(T*)

4-plex ○

6-plex ●

7-plex ■

○●■BTH274(C*)

○●■BTH274(C*)


(a)

BTH

274-

15a

EU

B33

8Ia-

23a

BFR

G60

2-19

a

C*T* G*

0

500

1000

1500

2000

2500

3000

3500

4000

4500

0 10 20 30 50 60 70 80 90

Size (nt)

Dye

inte

nsity

(rfu

)

BTT

1250

BA

C30

3-5a

BTH

274-

15a

40

● 6plex tube

(b)

0

2500

5000

7500

10000

12500

15000

17500

0 10 20 30 50

BIT

T141

BFG

1024

BA

DF1

037-

9a

BTH

274-

15a

BFR

G60

2-19

a

BU

FM10

18-1

8a

40

Dye

inte

nsity

(rfu

)

■ 7plex tube

Size (nt)

12


Quantifying relative abundance of specific targets in the influent and effluent from sewage treatment plant.

NDNDNDNDB. acidifaciens

NDNDNDNDB. intestinalis

NDND21.6±1.119.7±1.0B. uniformis

NDND5.1±1.24.9±0.7

BFRG602-related group

B. fragilis

NDNDNDNDBTT1250-related group

NDND0.3±0.30.5±0.1BTH274t-related group2

NDND0.8±0.21.1±0.1BTH274c-related group1

0.1±0.1ND3.6±0.54.5±0.4BFRG602-related group

1.1±0.91.6±0.110.0±0.311.1±1.4

Bacteria(EUB338Ia-23a)

Bacteroidales

Sample 2(n=3)

Sample 1(n=3)

Sample 2(n=3)

Sample 1(n=3)groupTarget

Effluent (%)Influent (%)Target abundance within a group

1 The group detected by primer extension with ddCTP of BTH274-15a.2 The group detected by primer extension with ddTTP of BTH274-15a.Calibration factors obtained for the 6-plexing, EUB338Ia-23a: BAC303-5a: BFRG602-19a: BTH274-15a(C): BTH274-15a(T): BTT1250=1: 6.1: 11.2: 14.4: 3.1: 2.4, and 7-plexing, BFRG602-19a: BUFM1018-18a: BTH274-15a(C): BTH274-15a(T): BFG1024: BITT141= 1: 0.8: 1.2: 0.4: 1.7: 0.1. Bacterial strains used include B. thetaiotaomicron, B. fragilis, B. acidifaciens (JCM10556), B. intestinalis (JCM13266), B. uniformis (JCM5828), Bacteroides. tectus (JCM10003), and Bacteroides pyogenes (JCM6294).ND, not detected.


Future (of) HOPE(s)

Targets- HOPE for RNA- HOPE for genomic DNA

Applications- HOPE for Bacteroides spp. (gut flora study)- HOPE for microbial source tracking- HOPE for quantification of nitrifying populations- many others more…

13


Gut flora

∆ Reaction one:Bth274 [T]_15dAPg_dtc732_20dABsls1016_10dABnd136Bth584_16dA

■ Reaction two:Bfrg602_19dABth274_15dABfrg1026_20dA

O Reaction three:Bfrg602_19dABcc1066_15dABadf1009Bitt141

● Reaction four:Bfrg602_19dABufm1018_20dABegt999Bhcg171_8dA

Reaction five:Bfrg602_19dABvg1016Bmsl1000_19dABcpc1015_24dA

# Reaction six:U1390_17dABac303_5dABfrg602_19dABdts_ gp980Bth274 [C]_15dABth274 [T]_15dA

Bovine-specific uncultivated Bacteroidales

Swine-specific uncultivated BacteroidalesCanines/Felines-specific uncultivated Bacteroidales0.02

∆ Pg_dtc732∆ Bsls1016∆ Bnd136

∆ Bth584

■ Bfrg1026

O Bcc1066O Badf1009

O Bitt141

● Bufm1018● Begt999

● Bhcg171

Bvg1016Bmsl1000

Bcpc1015

∆, # B

th274 [T]

■,O, ●,

, # Bfrg602

# Bac303

# U1390

# Bdts_gp980

Bacteroides tectusBacteroides pyogenesBacteroides salyersiaeBacteroides nordii

B. thetaiotaomicron


Bacteroides caccaeBacteroides acidifaciens

Bacteroides intestinalis

Bacteroides uniformisBacteroides eggerthii

Bacteroides helcogenes

Bacteroides vulgatusBacteroides massiliensis

Parabacteroides goldsteiniiParabacteroides distasonisParabacteroides merdae

Bacteroides coprocola

Other functionally important bacterial targets?Bifidobacterium spp.Clostridium leptum subgroupButyrate producing Firmicutes

Bacteroides and Parabacteroides spp.


H1

H2H3

P1, P2, P3Swine WW

C1, C2, C3

Municipal WW

Stress: 0

Human Pig CowLegend:

0 10 20 30 40 50 60

OthersB. fragilisB. caccae

B. acidifaciensB. intestinalisB. uniformisB. eggerthii

B. helcogenesB. vulgatus

B. massiliensisB. coprocola

Relative abundance (%)

B. fragilis cluster

Hong et al. AEM, 2008 (in press)

-10

0

10

20

30

40

50

MunicipalW

W

Sw

ine W

W

Pig

Cow

Hum

an

Others

B. fragilis clusterP. distasonis clusterB. fragilis subcluster [extends with ddCTP]

B. fragilis subcluster [extends wtih ddTTP]

Rel

ativ

e ab

unda

nce

(%)

14


Fecal source tracking

Fecal contamination in drinking water sources and recreational bodies

○Adverse health effects and liability issues

Conventional monitoring and detection○24 - 48h FC test ○Cultivation bias○Not able to pinpoint the fecal

origin


Human? Cow? Pig? Dog?

Host-specific primersPredominantly in human fecesBacteroides caccae (Bcc1066)Bacteroides fragilis (Bfrg1024)Bacteroides vulgatus (Bvg1016)Bacteroides uniformis (Bufm1018)

Bovine-specificUncultivated bovine-specificBacteroidales (C367)

Swine-specificUncultivated swine-specificBacteroidales (P163)

Canine-specificUncultivated canine-specificBacteroidales (D475)

1. Presence and absence of extended host-specific primer (library-independent)2. Relative abundance of each microbial target (library-dependent)

HOPE for microbial source tracking

15


Cow Pig Human Dog

C2C3C4C6C7C8

C9C10

C11

C12

C13C15C16C17C18C19

P1P2P3

P4 P5

P9P10 P11

P12

P13

P14

P16P17P18P19

H4H8

H10

H11

H12H13

H15H16

H17

H18

H19

H22

H23 H24

H25

H15N

D1

D2D4D5D6

D7

D8

D9

D10

D12

D13

D14

D18

c1

c2

c3c4

c5c6

c7

c8 c9

c10c11

p1p2

p3

p4p5p6p7

p8

h1

h2h3

h4

h5h6h7

h8

d1 d2d3

d4d5d6

d7

d8

Stress: 0.15

By establishing a HOPE-based library obtained from feces of known origin, a new sample contaminated with unknown sources can potentially be identified based on its location on this plot.

Hong et al. (unpublished)

Decide based on which type of host-specific primer extended


Thank You!

More HOPEs…

presentation:dr. liu, wen-tso · ast seminar in thailand, 11-12 mar 08 molecular biological tools...

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