Bacterial Genomics

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Bacterial Genomics
ACGTACGTACGTGCGTGAGATCGCTGATGCTGATCGTAGCTGATGCGCTA
AGATGATGATGCTGATGATGCTGATCGTAGTCGATGATCGTAGTCGATGC
TCGGCTGCGGGGCTGCTGGCTGCTGCTTATATATAGGATAGTAGATGATA
GTAGACTGATGCTGATCGTAGCTGATGCGCTAAGATGATGATGCTGATGA
TGCTGATCGTAGTCGATGAT

• Professor Mark Pallen
• University of Birmingham

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Bacterial Genomics

• What is it and why bother?
• First catch your genome
• Now you have a genome sequence
• 101 practical uses of a genome sequences
• The Future

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Genomic SpaceThe New Frontier
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The Post-Genomic Era

• Within the next five years, we will have the
  genome sequences
• of every significant bacterial pathogen of
  humans, plants and animals
• of all the model lab organisms, including the
  human genome

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What is bacterial genomics?

• Acquisition exploitation of whole genome
  sequences
• Think big!
• Study of 1000s of genes
• High-throughput
• Global approaches
• Automation and Technology-driven
• Catching the crumbs from the human genome project

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Why sequence genomes?

• Releases resources
• cheap
• only need do it once
• only way to get info
• renders problems finite
• new targets
• drugs, diagnosis, vaccines
• pilots for human genome
• because its there!

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The scale of the initiative

• gt200 bacterial genome projects underway
• Big players
• Wellcome Trust/Beowulf Genomics
• TIGR
• DOE
• NIH
• BBSRC

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Pathogen Sequencing at the Sanger Centre

• Bordetella pertussis
• B. bronchiseptica
• B. parapertussis
• Campylobacter jejuni
• Clostridium difficile
• Corynebacterium diphtheriae
• Mycobacterium tuberculosis
• M. bovis

• M. leprae
• N. meningitidis
• Salmonella typhi
• Staphylococcus aureus x2
• Streptococcus pyogenes
• Streptomyces coelicolor
• Yersinia pestis

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TIGR

• Archaeoglobus fulgidus
• Borrelia burgdorferi
• Deinococcus radiodurans
• Haemophilus influenzae
• Helicobacter pylori
• Methanococcus jannaschii
• Mycobacterium tuberculosis
• Mycoplasma genitalium
• Thermotoga maritima
• Treponema pallidum

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Others

• Escherichia coli
• Bacillus subtilis
• Saccharomyces cerevisiae

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First catch your genome...

• Choose strain
• Fresh isolate or lab strain?
• Generate template
• Small insert shotgun library
• Sequence
• ABI automated sequencers
• Assembly
• Closure and finishing
• Annotation

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Data release

• Information wants to be free!
• Immediate versus delayed release
• Powerful synergy with Internet and web
• Distance is dead!
• Distributed Resources
• User-friendliness of Web
• Just Cut and Paste!

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Genome Annotation

• Problems
• ORF identification
• Inferring function from homology
• Very labour-intensive
• Never complete
• need for ongoing curation

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Campylobacter jejuni NCTC 11168
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Campylobacter jejuni

• No pathogenicity islands, prophages, IS elements,
  retrons
• No new toxins, except for TlyA
• Evidence for a capsule?
• Polymorphic homopolymeric tracts
• Associated with gene clusters for surface
  structures
• Many variants present in same culture
• C. jejuni is a quasi-species!

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C. jejuni NCTC 11168
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Homopolymeric tracts
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Homopolymeric tracts
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Now you have a genome sequence.
Genome
Hypothesis Driven Research
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Complete Data Set

• Today we have naming of parts. Japonica
• Glistens like coral in all of the neighboring
  gardens,
• And today we have naming of parts
• "The Naming of Parts" by Henry Reed
• Fuelling hypothesis-driven research
• Hood et al. 1996 Mol Microbiol. 22951-65.
• Used genome sequence to unravel H. influenzae
  lipopolysaccharide biosynthesis

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Microarrays

• Arrange large number of hybridisation targets in
  a gridded array
• Variety of approaches
• Oligos
• Photomasking chip technology
• Affymetrix GeneChip
• Inkjet
• Amplicons
• Robotic spotting

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Microarrays

• Uses
• Global expression studies
• Global mutagenesis with molecular bar-coding
• Differential genomics
• Advantages
• Rapid, global survey of 100-1000s of genes
• Small scale
• saves reagents
• improved performance
• Spawn hypothesis-driven projects

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Global expression studies

• High-density gridded arrays
• PCR up each ORF
• Vector-primer PCR
• ORF-specific primers
• Grid out onto nylon filter or glass slide
• Make fluorophore or radio-labelled cDNA
• Hybridise to array
• look for differences in signal

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Global Mutagenesis

• Saccharomyces Genome Deletion Project
• knock out all ORFs
• simultaneously tag them with molecular barcode
• pool mutants
• stress
• amplify tags, hybridise to chip
• look for changes in hybridisation pattern
• Similar studies on bacteria (e.g. Bacillus
  subtilis)

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Proteomics

• Genome is thought, proteome action!
• Differential proteome analysis
• Confirmation correction of genome
• Transcription ? Expression

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Proteomics
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Proteomics

• Techniques
• 2D gels
• peptide mass fingerprinting (MALDI-TOF)
• peptide sequencing (nanospray)
• Example
• Salmonella periplasmic proteome
• Qi SY, et al. 1996 J Bacteriol. 1785032-8.
• O'Connor CD, et al. 1997 Electrophoresis.
  181483-90.

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Interaction Maps

• Protein-DNA interactions
• RNA-RNA and RNA-protein interactions
• Protein-Protein interactions
• Yeast and bacterial two-hybrid systems
• Protein contigs
• Global screens yeast and H. pylori

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Comparative Genomics

• Paradigms?
• Escherichia coli?
• A megabase more in some clinical isolates
• Helicobacter pylori?
• Largely stable
• PFGE samples biologically irrelevant changes

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The Genome of the Gaps

• Problem
• How typical are genome-sequenced strains of
  clinical isolates?
• What insertions, deletions and rearrangements
  occur in clinical isolates?
• Genomic core versus genomic options
• Prophages, PAIs, integrons, plasmids, transposons
• Solution
• Use genome sequence of sequenced strain(s) as
  starting point for exploring genomic diversity
• Microarrays
• Whole-genome PCR

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Whole-genome PCR

• 100-200 long PCRs
• 20-40 kbp amplicons
• Microtitre format
• High throughput
• Analyse amplicon patterns
• Variation in amplicon size
• Variation in pattern of drop-outs

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Infection GenomicsThe Battle of the Genomes
Human Genome
Microbial Genome
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Infection Genomics
Microbial Genome
Cell
Proteome
Transcriptome
Genetic basis of virulence
Cellular Microbiology
Protein-Protein Interactions
Differential Gene Expression
Cell
Genetic basis of host susceptibility
Proteome
Transcriptome
Human Genome
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Bacterial Genomics

• What is it and why bother?
• First catch your genome
• Now you have a genome sequence
• 101 practical uses of a genome sequences
• The Future

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The Gods Eye View?
or the Devils in the details?
Hypothesis Driven Research