Rapid taxonomic classification of complex consortia Rapid taxonomic classification of complex consortia of environmental rDNA using a microarray.of environmental rDNA using a microarray.

CEB - ESD - LBNLTodd DeSantis, Sonya Murray, Jordan Moberg, Gary Andersen

Carol Stone (DSTL, U.K.)

What bugs are in my What bugs are in my sample?sample?

The ponderings of a toddlerThe ponderings of a toddler

Why must Mom confiscate my “Hello Kitty”

blanket on laundry day?

Will the swings be wet at the park?

How will this sausage impact the

diversity in my lower G.I. bacterial

community?

Will I inhale any archaeal

microorganisms when I visit the

hot springs?Gianna DeSantis

• Every discarded water sample, geological core, or spent air filter is lost data.

• But who wants to do all the work?– Culture? Anaerobes? non-cultivable? Safety?– Analysis of nucleic acids isolated from environment

• Must classify or sort heterogeneous nucleic acids into bins.– Restriction Fragment Length Polymorphisms (RFLP)– Single Stranded Conformation Polymorphisms (SSCP)– Temp/Denat Gradient Gel Electrophoresis (T/DGGE)– Sequencing

» Provides taxonomic nomenclature » estimates the relative abundance » Need to create, clone, & process hundreds of samples

• Can we create a simple, comprehensive (quantitative?) microbial test?

Project OverviewProject Overview• Goal

– Create a single microarray capable of detecting and categorizing the bacteria in a complex sample.

• Approach– GeneChip targeted at

16S rDNA sequence variations to distinguish taxa.

The The RibosomeRibosome

rDNA

rRNA (functional molecule)

LSU

SSU16s or 18s

• Foundations:– Maintain the largest

16S gene library (~83,000).

– Cluster sequences into taxa (~8,000).

– Create algorithm for picking probes for each taxa (~500,000).

cctagcatgCattctgcatacctagcatgGattctgcata

MATCHMISMATCH

Build custom Affymetrix GeneChip

• Massive parallelism – up to 2 million probes upon a 1.28 cm2 glass surface.• Identification of multiple species in a mixed population•Pair each probe with a “mismatch” control probe.

General General ProtocolProtocol

Air

Soil

Feces

Blood

Water

rRNA

gDNA

Universal 16S rDNA

PCR

Contains probes adhered to glass surface in grid

pattern.

Overview of Sample Preparation using a Modified Affymetrix Protocol

18 µ

18 µ

AC

GG

TC

GA

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TC

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AC

GG

TC

GA

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TC

GA

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GG

TC

GA

Hybridize

PCR Amplify DNA

Fractionate DNA

End-label with biotin

Extract Genomic DNA

Progress in Miniaturization

GeneChip 3000 scanner 16S Microarray

500,000 Probe 16S array(DOE 16S Chip)

Parameter Frankia Clostridium Positive fraction 1.00 0.64Average difference 3720 625

Frankia sp. str. G48

PM MM

Clostridium butyricum

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BASIS

EPA

(127 OTUs displayed)

High confidence bacterial species detectionwith 500K probe microarray: - Greater microbial diversity in EPA samples

...CGTAAAGCTCTGTCTTTGGGGAAGATAATGACGGTACCCAAGGAGGAAGCCACGGCTAACT... C. perf. str.CPN50

................................................................... C. perf. resistant

................................................................... Clostridium sp. AB&J

................................................................... clone p-4636-2Wa2

................................................................... C. perf. A

................................................................... C. perf rrnA

................................................................... C. perf rrnE

.................................T................................. C. perf rrnD

................................................................... C. perf rrnC

................................................................... C. perf rrnB

................................................................... C. perf rrnF

................................................................... C. perf rrnG

................................................................... C. perf str.13a

................................................................... C. perf str.13b

................................................................... C. perf rrnH

................................................................... C. perf rrnI

................................................................... C. perf rrnJ

................................................................... clone OI1612

................................................................... C. perf. B

................................................................... Swine manure 37-3

................................................................... Swine manure 37-4

TAAAGCTCTGTCTTTGGGGAAGATA tacccaaggaggaagccacggctaa AAAGCTCTGTCTTTGGGGAAGATAA AAGCTCTGTCTTTGGGGAAGATAAT AGCTCTGTCTTTGGGGAAGATAATG

Bacteria

CFB

Cyan

Proteo

Gram +

High G+C

Bacil-Strep

Clostridium

C.BOTULINUM_SUBGROUP

C.THERMOBUTYRICUM_SUBGROUP

C.BARATI_SUBGROUP

C.CADAVERIS

C.ALGIDICARNIS

C.PERFRINGENS

C.AURANTIBUTYRICUM

C. BUTYRICUM

16S rDNA

Probes 5 - 8

5 6 7 8

27 1492

420 469

Ave Diff =1891

C. perfringens probe set identified in EPA sample 22 (N.Y.C., Spring)

-- ARCHAEA (1)---- EURYARCHAEOTA (1.1)------ METHANOMICROBACTERIA_AND_RELATIVES (1.1.3)-------- HALOPHILIC_ARCHAEA (1.1.3.4)---------- NTC.AMYLOLYTICUS_SUBGROUP (1.1.3.4.6)

(Natronococcus) 1.1.3.4.6.32228 OTU 6 seqs prokMSA_id:649 AB012049 str. MSP1 prokMSA_id:650 AB012052 str. MSP11 prokMSA_id:653 AB012055 str. MSP16prokMSA_id:654 AB012056 str. MSP17prokMSA_id:655 AB012057 str. MSP22 prokMSA_id:656 AB012058 str. MSP23

Developing Microarray Experiment Browser

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Beta protebacteria taxa

Sig

nal

(a.

u.)

434 -proteobacteria taxa

2250306000000.030_st Acidobacterium capsulatum+2280208040000.041_st Bordetella pertussis+2280209041300.025_st Acidovorax avenae+2280210040000.040_st Burkholderia graminis+2280210060000.007_st Burkholderia cepacia+2280211000000.071_st Oxalobacter formigenes+2280299000000.001_st Ralstonia solanacearum+2280308990000.001_st Stenotrophomonas maltophilia+

Clean Filter-extracted-amplified-fragmented-labeled-hybridized

Example of detection with the -proteobacteria Sub-division

Many of the detected taxa also contain "Environmental clone” members.

What’s in a “blank”

sample?

Testing the effects of variation in hybridization temperature

All 3 conditions allowed spikes to be detected.

48deg hybridization permitted the least additional taxa to be reported.

Create synthetic community of 16S amplicons.A. capsulatumY. pestisC. jejuniS. aureusB. anthracisS. typhiB. melitensisR. prowazekiiB. subtilisE. coli

Separately culture, gDNA-extract, and PCR-amplify.Combine PCR products.

Testing the effects of variation in hybridization temperature

All 3 conditions allowed spikes to be detected.

48deg hybridization permitted the least additional taxa to be reported.

Probe Set 45 hyb 48 hyb 50 hyb Members

2130500000000.006 _st+ + + RB25 (Nitrospina)

2131100000000.015 _st+ MB1228 (Nitrospina)

2131200000000.011 _st+ RB40 (Nitrospina)

2250306000000.030 _st+ + + Acidobacterium capsulatum

2280106990000.001 _st+ + + Brucella melitensis

2280108010199.001 _st+ + + Roseobacter litoralis

2280108050500.003 _st+ + + Rickettsia prowazekii

2280110990000.001 _st+ Sphingomonas yanoikuyae

2280205060000.005 _st+ Zoogloea DhA-35

2280209041400.009 _st+ + + Aquaspirillum metamorphum

2280320040000.039 _st+ + + Shewanella woodyi

2280323990000.001 _st+ + + Vibrio vulnificus

2280324020000.008 _st+ + + Aeromonas hydrophila

2280327040200.012 _st+ Erwinia amylovora

2280327050000.007 _st+ + + Klebsiella oxytoca

2280327070000.005 _st + Pantoea agglomerans

2280327090000.001 _st+ + Erwinia carotovora

2280327100000.011 _st+ Serratia marcescens

2280327120000.010 _st + Serratia proteamaculans

2280327140500.003 _st+ + + Yersinia pestis

2280327990000.002 _st+ + + E. coli, S. typhi

2280503040000.010 _st+ + + Campylobacter jejuni

2300107040000.027 _st+ + + R18 (Actinobacteria)

2300710090000.020 _st+ + Bacillus firmus

2300711050000.020 _st+ + + Bacillus subtilis

2300712010000.013 _st+ + + Staphylococcus aureus

2300712040000.004 _st+ + + Bacillus anthracis

2300717030000.017 _st+ + + Col7 (Lactobacilli)

2300721990000.001 _st+ + + Streptococcus

C. jejuni probe sets 45 deg C 48 deg C 50 deg C

2280503040000.010d_st 3015 2219 1927

2280503040000.010c_st 3011 2303 2126

2280503040000.010b_st 3166 2397 2548

2280503040000.010_st 3162 2320 2291

mean: 3088 2309 2223

standard deviation: 87 73 263

coefficient of variation: 0.03 0.03 0.12

Reproducibility

Probes for C. jejuni tiled in 4 areas

Columbus, OH

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Quantitative Considerations

-Spike environmental samples with defined amplicons.

- Calibrate

- Pearson’s corr coefficient was significant (df=18).

- 95% confidence intervals plotted.

Figure 2 - Calibration Plot

y = 0.9207x + 10.504R = 0.974

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log2 Concentration (pM)

log

2 H

ybS

core

Spike-in rDNA

Environmental rDNA

95% Confidence Limits

Spike-in Regression

Environmental community is measured with confidence intervals.

Figure 3 - Concentration of Environmental SSU Amplicons

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Clostridium thermobutyricumStreptococcus anginosus

Bacillus racemilacticusPseudomonas sp.

symbiont of Solemya velumClostridium limosum+

Eurotiales (Aspergillus+)Bartonella+

Staphylococcus delphini+Vibrio parahaemolyticus+

Pasteurella sp.Heterotextus alpinus

StreptomycesStaphylococcus cohnii+

Propionibacterium lymphophilumLeucostoma persoonii

Tax

a

rDNA Concentration (pM)

Conf Interval: Conc(t(RSE)/b)(1/m+1/n+((Y-y)2) / (b2(n-1)sx2))

b = slope from regression

Y = mean of 6 replicate measurements

m = number of repeat measurements = 6

y = mean of the HybScores for the 20 points used for calibration

t = critical value obtained from t-table for 18 d.f. for 95% = 1.734

RSE = residual standard error of calibration points = 0.56

sx = standard deviation of the conc. for the 20 points used for calibration

Current projectsCurrent projects

- Netherlands soil bioremediation tracking

- BioWatch – DHS metropolitan air microbe survey

- G.I. community comparison- Root-soil interface- Does polymerase brand affect perceived

community?

Takara PCR

Applied Bio PCR

PCR Enzyme Comparison

Biowatch gDNA

Biowatch gDNA

+

+

neg

neg

Takara (gDNA pool)Total ng DNA in PCR bands: 4437.58Total ng DNA in PCR reactions (+smear): 7266.95Ratio of bands:total DNA 0.61

Applied Bio. (gDNA pool)Total ng DNA in PCR bands: 546.73Total ng DNA in PCR reactions (+smear): 8149.28Ratio of bands:total DNA 0.07

ng in 5uL ng in 45uLLane 1 Band from biowatch pool (Tak) 76.3 686.9Lane 2 Band from biowatch pool (Tak) 59.3 533.8Lane 3 Band from biowatch pool (Tak) 80.5 724.9Lane 4 Band from biowatch pool (Tak) 99.2 892.8Lane 5 Band from biowatch pool (Tak) 104.3 938.5Lane 6 Band from pos control 1pg (Tak) 73.4 660.7Lane 1 Band+Smear from biowarch pool (Tak) 151.7 1365.3Lane 2 Band+Smear from biowarch pool (Tak) 146.8 1321.5Lane 3 Band+Smear from biowarch pool (Tak) 182.0 1638.4Lane 4 Band+Smear from biowarch pool (Tak) 161.3 1451.6Lane 5 Band+Smear from biowarch pool (Tak) 165.6 1490.1Lane 6 Band+Smear from pos control 1pg (Tak) 86.0 774.1Lane 9 Band from biowatch pool (AB) 14.1 127.0Lane 10 Band from biowatch pool (AB) 13.3 119.3Lane 11 Band from biowatch pool (AB) 7.8 70.4Lane 12 Band from biowatch pool (AB) 11.4 102.3Lane 13 Band from biowatch pool (AB) 14.2 127.7Lane 14 Band from pos control 1pg (AB) 8.3 74.4Lane 9 Band+Smear from biowarch pool (AB) 160.6 1445.1Lane 10 Band+Smear from biowarch pool (AB) 184.8 1663.6Lane 11 Band+Smear from biowarch pool (AB) 185.0 1665.2Lane 12 Band+Smear from biowarch pool (AB) 185.5 1669.9Lane 13 Band+Smear from biowarch pool (AB) 189.5 1705.5Lane 14 Band+Smear from pos control 1pg (AB) 132.3 1190.3

SummarySummary

The rDNA microarray is able to rapidly quantify and taxonomically classify complex consortia of rDNA amplicons.

You can collect data on over 8,000 taxa simultaneously.

AcknowledgementsAcknowledgements

• Gary Andersen – Group Leader • Carol Stone – UK sample collection, hybridization • Sonya Murray – sample preparation, hybridizations,

manuscript composition• Jordan Moberg – sample preparation, sample tracking

database, parameter optimization.