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Broad Resequences 3 TB Strains on Illumina GA; ‘Dozens’ to Follow in Diagnostic Hunt

In an effort to better understand tuberculosis drug resistance, researchers at the Broad Institute’s Microbial Sequencing Center have resequenced three Mycobacterium tuberculosis strains using both Solexa and Sanger sequencing.
The ultimate goal of the project, a collaboration between the Broad Institute, the Harvard School of Public Health, and the Nelson Mandela Medical School at the University of KwaZulu-Natal in South Africa, is to develop better diagnostics for TB drug resistance. To that end, the Broad team plans to sequence “many dozens” of additional tuberculosis strains, ideally using only next-generation technologies, according to a project participant.
The scientists, who recently released their analysis on the web, prior to publication in a peer-reviewed journal, sequenced three clinical isolates of strains from a recent TB outbreak in KawaZulu-Natal: a drug sensitive one, a multiple drug resistant one, and an extreme drug resistant one.
Initially, they sequenced these KZN strains using Illumina’s Genome Analyzer, with a coverage of about 30X, and aligned these reads to an outgroup reference strain in order to identify polymorphisms.
But “because it’s a fairly new technology, we wanted to make sure that we were getting accurate results,” James Galagan, associate director of microbial genome analysis at the Broad Institute, told In Sequence last week. To check their results, the scientists also analyzed the same three strains by deep-coverage Sanger sequencing.
After assembling the Sanger reads, they compared the results to the Illumina data. “It was a way of making sure that the SNPs we got were not an artifact of Solexa sequencing,” Galagan explained. They were, in fact, able to confirm all the SNPs discovered by the Illumina platform with the Sanger data.
However, the Illumina platform missed “a handful of other mutations,” such as insertions, deletions, and transposons, that the researchers were able to detect in the assembled Sanger data, according to Galagan.
The Illumina data also did not cover a number of repeat-rich genes where the short reads from the next-gen platform did not align uniquely. “Part of this is not necessarily the Solexa sequencing that was the problem, it was the stringency by which we aligned the Solexa data back to the reference genome,” Galagan explained.
Overall, the researchers were happy with the quality of the Illumina data, but they are still looking for improvements. “We did not miss enough [mutations] that it made us worry about the results we got from Solexa, but I still think it’s fair to say that it’s early days for using these technologies, and so we continue to evaluate [them],” Galagan said.
“The hope would be that these new sequencing technologies will eventually be the only solution we need. But the ultimate shape of that solution is still being very much investigated.”
The researchers decided to use Illumina’s platform for this project because it had given them good results in a pilot project, where they used the technology to resequence F11, a TB strain they had previously sequenced and finished using Sanger technology (see In Sequence 9/4/2007). Following that pilot, “we knew that we had very good sensitivity in terms of resequencing with Solexa and identifying polymorphisms,” Galagan said. “Because we got such good results for Solexa in our proof-of-principle, we decided to go with Solexa.”
454 in South Africa
Galagan’s colleagues in South Africa, on the other hand, decided to use 454’s sequencing platform to sequence another extremely drug resistant KZN strain. Comparing the two data sets, Galagan said, “will be an interesting comparison, certainly.”

“The hope would be that these new sequencing technologies will eventually be the only solution we need. But the ultimate shape of that solution is still being very much investigated.”

The reason the South African researchers chose 454’s platform, which is housed at the Nelson Mandela School of Medicine at the University of KwaZulu-Natal, “is simply its availability,” Willem Sturm, dean of the medical school, told In Sequence by e-mail.
The sequencer, which was funded by the South African government through an organization called Lifelab, is housed at the medical school, and “the acquisition of this equipment coincided with the emergence of the XDR epidemic, and it made sense to ‘inaugurate’ the equipment by sequencing one of the many isolates we have,” he said. Sturm and his colleagues have not published their results yet and did not say when they are planning to do so.
The Broad researchers found that only a few polymorphisms differentiate the drug sensitive, the MDR, and the XDR strains. But in order to comprehensively characterize mutations associated with drug resistance, they will need to sequence many more strains from different geographic regions and phylogenetic origins. “Certainly we want to sequence many dozens,” Galagan said.
For now, the Broad team will continue to use Illumina’s Genome Analyzer, but Galagan did not exclude using other technologies in the future. “We are always carefully evaluating the data we get, and if it makes sense to incorporate other technologies, then we would certainly do that as well,” he said.
After compiling drug-resistance mutations, the researchers plan to validate these in other drug-resistant strains, and ultimately use them as the basis for a molecular diagnostic test for drug resistance that could be hybridization-based or PCR-based.
The goal for a diagnostic, Galagan said, is to choose a technology “that can be utilized in the field, or if not in the field, at least in endemic areas where they don’t necessarily have to have a large sequencing facility.”

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