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Researchers Test Pyrosequencing Approach for Characterizing Potential Outbreak Bacteria

NEW YORK (GenomeWeb News) – Next-generation sequencing and comparative analysis are effective ways to obtain and analyze bacterial sequence data quickly, new research suggests — something that may be necessary in the event that bacteria are used as biological weapons.
In a paper that appeared online today in the journal Genome Research, French and Swedish researchers used the GS20 sequencing platform from Roche’s 454 Life Sciences to sequence and compare virulence and resistance factors in Francisella tularensis subspecies holarctica — a bacteria that’s considered a potential bioterrorist threat — in a matter of weeks.
“With this technique it’s possible now to obtain the complete sequence of a bacteria in just six weeks time,” lead author Bernard La Scola, a bacteriologist at the University of the Mediterranean in Marseille, told GenomeWeb Daily News
F. tularensis is an extremely infectious bug that often occurs naturally in animals such as rodents and rabbits. In humans, it can cause tularemia, a condition characterized by a sudden fever, chills, dry cough, weakness, muscle aches, joint pain, and diarrhea. Depending on the type of exposure, tularemia may also involve skin or mouth ulcers, swollen lymph glands, and a sore throat. It may eventually progress to bacterial pneumonia.
Consequently, many worry that the bacteria could be manipulated and used as a biological weapon. “It’s considered one of the three high-risk bacteria in bioterrorism,” La Scola said. “You can introduce a resistance gene or you can cultivate it on an antibiotic and select resistance — and it’s not difficult to do.”
In such an event, La Scola explained, having the ability to sequence an outbreak strain as quickly as possible may be critical. While it’s possible to detect antibiotic resistance without sequencing, the sequence data can reveal the mechanism of resistance. It also can help researchers track a strain’s origin.
To determine whether pyrosequencing was an efficient way to generate this sequence data, La Scola and his colleagues used two GS20 runs to generate 1,800,530 base pairs of sequence from an F. tularensis subspecies holarctica strain called URFT1 to an average depth of coverage of 26 times.
However, while acquiring sequence data is relatively fast, taking only about a day, the analysis is more time consuming, which is why the researchers reported a six-week turnaround. 
Nevertheless, La Scola said he expects the approach will likely take even less time as newer and faster sequence analysis software is developed. “The difficult part is to analyze the sequences,” he said.
Still, he explained, pyrosequencing shaves time off of the process. For instance, he explained, sequencing the entire F. tularensis genome by Sanger sequencing could take two or three months.
The team also further streamlined the process: instead of sequencing and assembling the entire genome, they decided to forego the finishing process. The researchers then assembled 480 URFT1 contigs and compared these with sequence from other known F. tularensis strains.
As it turned out, most of the remaining sequence gaps fell within repeat regions. Because these repeat sequences don’t contain information about virulence factors or other genes of interest, La Scola explained, sequencing them is unnecessary in the outbreak context.
Indeed, La Scola and his colleagues detected various polymorphisms, modifications, and deletions that could infer changes in characteristics such as virulence and antibiotic resistance. They also used the information they gleaned to develop a typing system that allowed them to categorize dozens of F. tularensis holarctica strains, including URFT1; a live vaccine strain; 74 Swedish, clinical isolates collected over ten years; and five clinical specimens.
“In the bioterrorism context, it allows the rapid detection of strain manipulation, including intentionally added virulence genes and genes that support antibiotic resistance,” the authors wrote.

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