Rapid taxonomic classification of complex Rapid taxonomic classification of complex consortia of environmental rDNA using a consortia of environmental rDNA using a microarray. microarray. CEB - ESD - LBNL Todd DeSantis, Sonya Murray, Jordan Moberg, Gary Andersen Carol Stone (DSTL, U.K.) What bugs are in my What bugs are in my sample? sample?
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Rapid taxonomic classification of complex consortia of environmental rDNA using a microarray. CEB - ESD - LBNL Todd DeSantis, Sonya Murray, Jordan Moberg,
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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
(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
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