Optical Mapping of E-coli O157:H7
Alex Lim
Laboratory for Molecular and Computational
Genomics
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
Laboratory for Molecular and Computational
Genomics
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
Laboratory for Molecular and Computational
Genomics
7 terabytes
of raided
storage!
Laboratory for Molecular and Computational
Genomics
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
Laboratory for Molecular and Computational
Genomics
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
Laboratory for Molecular and Computational
Genomics
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 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
Genomic DNA molecule from E.coli O157
Laboratory for Molecular and Computational
Genomics
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
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
Laboratory for Molecular and Computational
Genomics
A comprehensive overview of OM accuracy
Laboratory for Molecular and Computational
Genomics