DOE Genomics: GTL

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DOE Genomics GTL Annual Retreat
2005 Sequence-Based Discovery Carl
Abulencia Diversa Corporation San Diego,
California
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Sequence-Based Discovery

• Goals
• Gain a better understanding of how organisms live
  in contaminated environments, and how they have
  adapted to external stressors.
• Obtain sequence data by accessing the genomes
  from the organisms living in contaminated
  environments.
• Analyze the genes, operons, and stress response
  pathways from the natural diversity to study
  evolutionary changes, selections, expansions and
  gene transfers.

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Technology Flow for Sequence-Based Discovery
Contaminated Soil
Microbial DNA
Extract large- fragment DNA
MDA amplify clone
Screen for genes involved in stress response
Form Genomic Libraries
Pathways analyzed by computational core
Genes and Pathways Analyzed by Functional Genomics
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Samples From the NABIR FRC SiteOak Ridge, Tenn.

• FRC Sampling, November 2004
• 9 uranium contaminated soil samples and one
  background sample.
• 12 Large fragment DNA extractions completed on 9
  samples.

• Limitations of sampling from contaminated soil
  sites
• Very low biomass.
• Very little extracted DNA.
• DNA concentrations too low for
• 16S rRNA gene PCR
• DNA Library construction

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Amplification of Sample DNAWhole Genome
Amplification
Multiple Displacement Amplification (MDA)

• Isothermal amplification method.
• phi29 polymerase.
• Processive (up to 70kb).
• Strand-displacing.
• 3-5 exonuclease activity (proofreading
  activity).
• Random hexamer primers.
• 3 hour 16 hour reaction time.

97kb
49kb
12kb
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MDA Genome Coverage Analysis
Affymetrix E.coli Genome GeneChip Array

• 7123 probe sets on the chip that represent the
  full genome.
• Presence or absence of 700 bp regions of sample
  DNA are detected by DNA hybridization.

• Comparison of Unamplified to Amplified gDNA
• Positive Control is gDNA from an overnight
  culture (unamplified).
• Dilute culture to 5000 cells, extract gDNA, MDA
  amplify.
• Dilute culture to 5 cells, extract gDNA, MDA
  amplify .

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MDA Bias Analysis

• Mix the isolate gDNAs.
• Create a library from unamplified,mixed DNA.
• (66 µl of 60ng/ µl 4µg)
• Create two libraries from MDA amplified, mixed
  DNA.
• amplified from 1/100 dilution of mixed DNA.
  (600pg)
• amplified from 1/10,000 dilution of mixed DNA.
  (6pg)

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Sequence Analysis of Random Library Clones
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From Soil to Sequence

• MDA
• 16S rDNA Libraries
• DNA Libraries

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16S rDNA Library Sequence Data
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Library Clone QC Sequencing Data
Library 3868, Sample 1, Area 3,
FB-075-04-07 Depth 324-360 inches
Library 3875, Sample 3, Area 3,
FB-075-04-07 Depth 144-180 inches
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Interesting Library QC Sequences
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Library Screening for Histidine Kinase Genes

• Histidine kinase superfamily chosen for
  sequence-based discovery from environmental
  libraries.
• Cells sense and respond to extracellular stimuli
  through phosphotransfer-mediated signaling
  pathways controlled by HKs and response
  regulators.
• Library clones containing HKs retrieved by DNA
  hybridization using degenerate probes designed
  from a subfamily of HKs from D. vulgaris.

Sense Primer 5-GGCSCAYGARATSAACAACCC-3
5-GGTSGTGAAGAASGGYTCGAA-3 Antisense Primer
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A Histidine Kinase Library Clone
two-component system sensor histidine kinase
malate dehydrogenase
ORF 3
ORF 2
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Library Clone 3870PT9 Sample 1, Area 3 3890bp
Partial ORF
ORF 1
outer membrane lipoprotein
two-component system response regulator
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Histidine Kinase Discovery Progress

• Sequence-Based Screening
• 12 Libraries Screened, 50,000 clones/ library
• 26 HK clones in Sequencing

• Library QC sequencing
• 34 HK clones fully sequenced

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Summary

• gDNA extracted from 9 contaminated soil samples
• MDA used to amplify gDNA from low-yield samples
• 16S rDNA libraries constructed from each sample
• DNA libraries constructed from each sample
• Library diversity verified by random QC
  sequencing
• Histidine kinases discovered in libraries by
  random sequencing
• Desulfovibrio-like histidine kinases discovered
  in libraries by sequence-based hybridization
• 16S rDNA sequences, library QC sequences, and
  full length HK clone sequences all uploaded to
  the VIMSS database
• A manuscript for publication is near completion
  detailing analysis of DNA libraries constructed
  from contaminated environmental soil samples

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Acknowledgments

• LBNL Dominique Joyner, Sharon Borglin, Eoin
  Brodie, Terry Hazen
• LBNL Eric Alm, Adam Arkin
• LBNL Tamas Torok
• ORNL David Watson
• Diversa Denise Wyborski, Mircea Podar, Joe
  Garcia, Cathy Chang, Sequencing Group, Keiko
  Obokata, Toby Richardson, Sherman Chang, Karsten
  Zengler, Martin Keller