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HMP Researcher Offers Insights into Shotgun Sequencing of GI Bugs

PHILADELPHIA (GenomeWeb News) – Preliminary high-throughput shotgun sequencing analyses of microbial communities in the human gastrointestinal tract are shedding light on the type and amount of sequence data needed to glean information from such experiments.

During a presentation at the American Society for Microbiology conference earlier this week, George Weinstock, associate director of Washington University's Genome Sequencing Center, outlined his group's efforts to use high-throughput shotgun sequencing to characterize microbial communities in the gastrointestinal tract.

The team is evaluating the approach as part of the Human Microbiome Project, a National Institutes of Health Roadmap project aimed at characterizing the microbes on and in the human body.

While much of the microbial community sampling so far has relied on 16S rRNA sequences to identify microbes associated with different parts of the human body, researchers are looking towards additional strategies for understanding such communities on a deeper level.

The researchers obtained fecal samples from three unrelated individuals and sequenced microbial communities in the samples using Roche 454 and Illumina platforms, Weinstock said. After obtaining billions of bases of DNA sequence data with each platform, they then compared their high-throughput data with information obtained using other techniques and compared the platforms with one another.

For instance, Weinstock said that by looking at how well reads from one platform covered reads from the other, they could get an idea of how exhaustively each approach sampled the microbial community. He noted that results so far indicate that Illumina sequencing provides fairly exhaustive sampling of the microbial communities tested.

By looking at the 16S rRNA sequences and validating these findings by other approaches, the team detected and confirmed thousands of known bacterial 16S rRNA sequences as well as dozens of yet-unclassified 16S sequences.

The researchers also estimated how much sequence data they'll need to see rare microbes in GI microbial communities by aligning shotgun sequencing reads to finished genomes of microbes known to be present in GI microbial communities — including Bacteroides thetaiotaomicron, which is commonly found in the intestinal tract, and the much rarer bug Escherichia coli. That work suggested that sequencing roughly ten billion bases will reliably sample rare organisms in these communities with the current technology.

While most of the shotgun sequencing reads corresponded with bacterial sequences, Weinstock noted that the team also ran across sequences from eukaryotes such as the protist Plasmodium, the green algae Chlamadymonas, and the fungus Aspergillus with Roche 454 sequencing. Compared with Sanger sequencing, Weinstock added, 454 sequencing also appears to be promising way to detect new viruses in the GI tract.

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