Second generation DNA sequencing on this level presents a formidable informatics challenge. The Human Microbiome Project has taken a metagenomic approach to identifying the bacteria in a wide variety of sites on and in the human body because the substantial majority of these bacteria have not been grown in culture. The correlations among the assays were excellent. Overall, we have employed 192 molecular probes representing 40 bacteria to detect the bacteria in twenty-one vaginal swabs as assessed by the Affymetrix GenFlex Tag16K assay and fourteen of those by the Sequencing by Oligonucleotide Ligation and Detection assay. By adding one unique oligonucleotide barcode for each clinical sample, we combine the samples after processing, but before sequencing, and sequence them together. To multiplex the clinical samples, we introduce a second, independent assay for the molecular probes employing Sequencing by Oligonucleotide Ligation and Detection. While the assay on Affymetrix GenFlex Tag16K arrays allows the multiplexing of the detection of the bacteria in each clinical sample, one Affymetrix GenFlex Tag16K array must be used for each clinical sample. In this communication, we report the first results of employing our molecular probes to detect bacteria in clinical samples. Rather, the molecular probe technology requires only a sequence of forty sequential bases unique to the genome of the bacterium of interest. This molecular probe technology does not require growth of the bacteria in culture. To that end, we adapted molecular inversion probes to detect bacteria using solely a massively multiplex molecular technology. Our ultimate goal is to detect the entire human microbiome, in health and in disease, in a single reaction tube, and employing only commercially available reagents.
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