Scientists find new markers for anthrax isolates

In a pioneering use of genomics as a tool for the forensic analysis of microbes, scientists at The Institute for Genomic Research (TIGR) in Rockville, Md., and at Northern Arizona University in Flagstaff, Az., have found new genetic markers that distinguish the Bacillus anthracis isolate that was used in last fall’s bioterror attack in Boca Raton, Florida, from closely related anthrax strains. Their findings, posted on Science Express on May 9 and scheduled for later publication in Science, demonstrate for the first time that the analysis of the genomes of microbial pathogens can be an effective method of finding new “genetic fingerprints” that can help trace the differences among almost identical strains of microbes such as anthrax. Previous genetic-marker analysis had focused on a limited number of DNA segments, rather than the entire genomic sequence. Citing those findings, TIGR President Claire M. Fraser called for the development of a comprehensive database of genomic analyses that could help investigators quickly pinpoint the sources of many different strains of anthrax and other potential biological warfare agents. That, in turn, would help scientists track down the source of the microbes and also help researchers develop better vaccines or medical treatments to counter the pathogens. Fraser is a member of a National Research Council panel that is drawing up a report about how scientific research can help counter bioterrorism. “Genome-based analysis will provide a powerful new tool for investigating unexpected disease outbreaks – whether they are bioterrorism attacks or natural outbreaks of more familiar pathogens,” said Fraser. A grant from the National Science Foundation (NSF) funded the sequencing of the Florida anthrax isolate, which TIGR compared to its nearly-completed sequence of another Ames-strain isolate obtained from Porton Down, U.K. The Porton sequencing project, begun in 1999, was funded by grants from the U.S. Office of Naval Research, the National Institute of Allergy and Infectious Diseases (NIAID), the Department of Energy and the United Kingdom’s Defense Sciences Technology Laboratory.

TIGR’s main anthrax researcher, Timothy Read, said this week that his group plans to sequence at least 14 other strains and isolates of B. anthracis over the next year or so in conjunction with the University of Northern Arizona laboratory led by microbiologist Paul Keim that has a library of about 1,200 anthrax isolates. That new anthrax sequencing project is funded by the NIAID.

“To lay the groundwork for future investigations, genome-based surveys of natural variation in all major pathogens, not just potential biological warfare agents, should be undertaken,” the scientists write in their paper. “Building a comprehensive database of information related to gene content, variant SNPs, indels and inversions in the genomes of important pathogens will allow investigators to quickly pinpoint the isolate that is most closely related to an outbreak strain. Such a database would greatly accelerate investigations and may deter future attacks.”

Analyzing the Isolate

In the Science Express paper, TIGR scientists – led by Fraser, Read, and TIGR’s chief of bioinformatics, Steven Salzberg -- report that the comprehensive comparison of the Porton and “Florida” anthrax isolates identified slight differences in their DNA that could be used as genomic fingerprints in future analyses.

Those differences include what the scientists describe as four “high-quality” single-nucleotide polymorphisms (SNPs) – differences of a single DNA base – between the sequenced chromosomes of the Porton and Florida isolates. In all, the comparison revealed 60 new genomic “markers” which could be useful in distinguishing among various anthrax isolates. Most of those differences were found in the plasmids – circular units of DNA that are separate from the chromosomes that contain most of the organism’s genetic material.

To evaluate whether those newly-found genomic differences are useful genetic markers to discriminate between anthrax strains, TIGR worked with Keim to use the new markers to analyze 6 anthrax isolates that initially had appeared to be indistinguishable from the original Ames strain based on existing genotype information. The new markers allowed the scientists to divide the 6 Ames isolates into 4 different categories.

TIGR shared its data with scientists from the Federal Bureau of Investigation (FBI) who are investigating last fall’s anthrax mailings, but Fraser said the TIGR research project was not part of that investigation. She said the FBI had placed no restrictions on TIGR’s publication of its results in a scientific journal.

To support the claims made in its paper, as well as to make it possible for outside researchers to search for additional differences in the genomic sequence of B. anthracis , TIGR has posted sets of anthrax data on its website (www.tigr.org): 1) the set of all assembled “contigs” (contiguous small segments of the genome) of all sizes; 2) the set of individual sequences that were used to create those contigs; 3) a file containing the quality values (estimates of error rates) for each individual sequence; and 4) a set of 1,000-base pair regions centered on each SNP to allow other scientists to identify and test the markers on other anthrax strains. Sequence data from the B. anthracis research also have been placed in the Genbank repository.

Read and his team plan to publish the full sequence of the B. anthracis genome in a scientific journal later this year. “The genomic sequence will boost the efforts to develop new vaccines, drugs and detection methods,” Read said. He added that the next wave of TIGR anthrax research “will help make B. anthracis a model for how genomics can be used to study bacterial pathogens.”

The Institute for Genomic Research (TIGR) is a not-for-profit research institute based in Rockville, Maryland. TIGR, which sequenced the first complete genome of a free-living organism in 1995, has been at the forefront of the genomic revolution since the institute was founded in 1992. TIGR conducts research involving the structural, functional, and comparative analysis of genomes and gene products in viruses, bacteria, archaea, and eukaryotes – higher animals and plants.

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