Optical Mapping of Ecoli O157:H7

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Optical Mapping of E-coli O157H7
Alex Lim
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Physical Maps

• Determine the relative position of landmarks
• along a genome segment

• Used as a basis for DNA sequencing and for
• the isolation and characterization of genes

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How Optical Mapping Works
DNA molecule is extended and aligned on surface.
Restriction enzymes cut the DNA molecule into
fragments.
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7 terabytes of raided storage!
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• Optical Mapping
• Uses fluorescence microscopy to create physical
  maps by directly imaging individual surface bound
  DNA molecules after cleavage by restriction
  enzymes
• Advantages
• solves the problem of determining fragment order
• small amounts of starting material
• easy to automate and high throughput
• offers information of the structural organization
  of genomes

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Single Molecule Optical Maps are Imperfect
Four major errors 1. False negatives (missing
restriction sites as a result of partial
digestion) 2. False positives (spurious cuts
not from enzymatic actions) 3. Sizing
errors 4. Loss of small fragments
These issues are dealt with by Gentig
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Optical Mapping of E. coli O157

• Produces shiga like toxin
• Causes hemolytic uremic syndrome (HUS) and
• hemorrhagic colitis
• Causes over 100,000 illness anually in US
• Sequenced by Blattners Group
• Perna, N.T., et al., Genome sequence of
  enterohaemorrhagic Escherichia coli O157H7.
  Nature, 2001.
• 409(6819) p. 529-33

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Genomic DNA molecule from E.coli O157
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E. coli O157H7

• 50x coverage
• Mapped with Xho I and Nhe I
• Very little editing required due to system and
  algorithmic improvements

Sequence
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Results of E. coli O157

• A total of 494 molecules of O157 genomic DNA were
  analyzed
• 251 molecules went into the final contig, 58 by
  mass,
• providing 30? coverage
• 166 Mb of total DNA analyzed
• Average fragment size generated by XhoI
• 25.1 kb
• The genome size was calculated to be 5.53 Mb

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A comprehensive overview of OM accuracy