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Optical maps guide shotgun sequencing from start to finish
Edward R. Winstead

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Twenty microbial genomes are scheduled to be sequenced in just one month at a laboratory in California early next year. Scientists at the Joint Genome Institute in Walnut Creek will use shotgun-sequencing combined with "optical mapping" to pull off this tour-de-force, which, if it works, will help researchers in laboratories around the world.

Detail of whole genome XhoI restriction map of E. coli O157 generated by shotgun optical mapping. View full

"Optical mapping is an advanced reconnaissance technique that tells us what an unknown genome looks like," says Daniel Drell, of the Microbial Genome Program at the US Department of Energy, which funds the institute in Walnut Creek. "It provides useful information you want to have before you begin, such as the size of the genome, and how many pieces it comes in."

‘Optical mapping is an advanced reconnaissance technique that tells us what an unknown genome looks like.’

In preparation for this sequencing event, DNA from some of the different microbes has been shipped to David C. Schwartz at the University of Wisconsin-Madison, who will do the optical mapping. "We're like the genomic Marines dropping in," says Schwartz, "because not much is known about a lot of these organisms. Our job is to deliver maps to be used in the assembly and to help verify that the sequence is correct."

Computer scientists, chemists, engineers, and biologists have worked with Schwartz to develop the techniques and tools for analyzing genomes visually. Two optical maps were used during the recent sequencing of E. coli O157:H7, the strain known to cause sometimes fatal food poisoning. The maps are in this month's issue of Genome Research.

Optical maps are created independently and without any sequence data. Their potential value was demonstrated three years ago, when a map revealed unexpected chromosomes in Deinococcus radiodurans. This microbe can survive a thousand times more radiation than humans can. Large doses of radiation shatter the microbe's DNA, but it can stitch the broken strands back together in about a day.

The Institute for Genomic Research (TIGR) in Rockville, Maryland, sequenced D. radiodurans in 1999 to learn about its DNA repair genes. As the assembly neared completion, the researchers turned to the optical map to verify the work. The map indicated that D. radiodurans has four chromosomes rather than the single circular chromosomes seen in other microbes. The new architectural information made sense to the assembly team and facilitated the ordering of some hard-to-place fragments.

Optical mapping of D. radiodurans genomic DNA with Nhe I. View larger

"The map certainly sped up the de-bugging phase that has to happen in every assembly project," says Owen White of TIGR. "Optical mapping works well as a way to verify where the assembly is working and where more attention is needed. It's a confidence builder." The complete D. radiodurans genome was published in Science in September 1999.

Two months later, Schwartz and colleagues published an optical map of the human malaria parasite. The map is used by the consortium sequencing the parasite to verify the assembly of all 14 Plasmodium falciparum chromosomes. The consortium includes Stanford University in California, the Sanger Centre in Britain, the Naval Medical Research Center, and TIGR.

The decision to undertake the genome sequencing was based in part on the successful assembly of chromosome 2, which was confirmed by optical mapping data.

Early on, some members of the malaria research community doubted that the genome sequence could be determined. The parasite's DNA is unstable, so whole-genome shotgun sequencing—which involves small DNA fragments—seemed to be the only viable method. But large regions of the P. falciparum genome are long runs of A's and T's. These regions, some researchers said, would confuse the computer assembly programs and make the project impossible to finish. The completion of chromosome 2 persuaded the consortium to undertake the genome project, and Schwartz's laboratory mapped the remaining chromosomes.

Detail from schematic of shotgun optical mapping. View full

"Being able to verify that we assembled chromosome 2 correctly from the optical map certainly gave us confidence to move forward and do the complete genome," says Daniel J. Carucci, Director of the Malaria Program at the Naval Medical Research Center in Maryland. "The optical maps provided a completely independent and highly verifiable way of assuring that computers can do a good job of assembling genomes."

The consortium plans to publish the complete P. falciparum genome with annotation in 2002.

The sequencing of E. coli O157:H7 was done at the University of Wisconsin-Madison, and the researchers, led by Frederick Blattner, encountered difficult sections of the genome. To provide a higher resolution map, Schwartz's laboratory combined two maps made with different restriction enzymes, which cut DNA molecules into fragments at specific sequences that are known. The DNA was labeled with fluorescent dye and the intensity of the glow correlated with the length of a fragment.

"Fragments twice as long as other fragments give off twice as much light," says Alex Lim, who led the optical mapping of E. coli at the University of Wisconsin. The two composite maps were combined into one optical map representing about 800 images of the E. coli genome. Blattner's laboratory compared the map to their data, and the final sequence was published in Nature last January.

One of the microbes Schwartz will soon be mapping for a Walnut Creek project in future years is Thalassiosira pseudonana. Climate researchers are interested in the marine organism because it plays a role in regulating carbon dioxide concentrations in Earth's environment. Oceans lower atmospheric levels of 'greenhouse' gases by absorbing carbon dioxide and storing carbon.

In addition to preparing for the microbial sequencing project, Schwartz's team is currently creating optical maps of the rice and human genomes. The human data will be used to help close gaps that remain in the nearly complete human genome sequence.

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Lim, A. et al. Shotgun optical maps of the whole Escherichia coli O157:H7 genome. Genome Res 11, 1584-1593 (September 2001).
Perna, N.T. et al. Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409, 529-533 (January 25, 2001).
Lai, Z. et al. A shotgun optical map of the entire Plasmodium falciparum genome. Nat Genet 23, 309-313 (November 1999).
Lin, J. et al. Whole-genome shotgun optical mapping of Deinococcus radiodurans. Science 285, 1558-1562 (September 1999).
Jing, J. et al. Optical mapping of Plasmodium falciparum chromosome 2. Genome Res 9, 175-181 (February 1999).

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