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Optical Mapping of E-coli O157:H7

Alex Lim

Laboratory for Molecular and Computational Genomics

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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

Laboratory for Molecular and Computational Genomics

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Laboratory for Molecular and Computational Genomics

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How Optical Mapping Works

DNA molecule is extended and aligned on surface.

Restriction enzymes cut the DNA molecule into fragments.

Laboratory for Molecular and Computational Genomics

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Laboratory for Molecular and Computational Genomics

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7 terabytes of raided storage!

Laboratory for Molecular and Computational Genomics

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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

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    • 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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Laboratory for Molecular and Computational Genomics

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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

Laboratory for Molecular and Computational Genomics

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Laboratory for Molecular and Computational Genomics

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Optical Mapping of E. coli O157

  • Produces shiga like toxin

Causes hemolytic uremic syndrome (HUS) and

hemorrhagic colitis

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  • Causes over 100,000 illness anually in US

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  • Sequenced by Blattner’s Group

Perna, N.T., et al., Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature, 2001.

409(6819): p. 529-33

Laboratory for Molecular and Computational Genomics

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Genomic DNA molecule from E.coli O157

Laboratory for Molecular and Computational Genomics

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E. coli O157:H7

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

Sequence

Laboratory for Molecular and Computational Genomics

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Results of E. coli O157

  • A total of 494 molecules of O157 genomic DNA were analyzed

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  • 251 molecules went into the final contig, 58% by mass,

providing 30× coverage

    • 166 Mb of total DNA analyzed

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  • Average fragment size generated by XhoI

25.1 kb

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  • The genome size was calculated to be 5.53 Mb

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Laboratory for Molecular and Computational Genomics

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A comprehensive overview of OM accuracy

Laboratory for Molecular and Computational Genomics