Bacterial Identification Single Pathogen Identification by 16S rRNA ...

9/13/2012• Organisms must compete to be isolated frominitial culture (low abundance organisms willbe missed).• Due to time and cost, a limited number ofgrowth conditions are routinely utilized.• Unculturable organisms‐ unknown preferredgrowth conditions or novel organisms• Focus is on identifying pathogenic organismsrather than assessing the complete bacterialcommunity.Aggregate DataQuality Filter Assess match/mismatch to MIDs, primers Remove low quality/ambiguous base calls Remove sequences that are too short/long Minimize sequencing “noise”Remove chimeric sequencesIdentify OTUsAssign identities to OTUs/sequencesCompare among/between communities• Put a “name” on it…definingoperational taxonomic units (OTUs)• Numerical “species”• 16S rRNA genes, sequences sharing >97%similarity• Application of classical ecological metrics• Comparing composition amongcommunities• Shared vs. “unique” taxa• Differing proportions of those taxa• Measuring diversity• Alpha diversity (withinsample/subject/group)• Beta diversity (betweensamples/subjects/group)• Baseline of“healthy” adults• 18 different bodysites sampled• Standardization of• Specimen collection• Processing• Sequencing• AnalysisC Huttenhower et al. Nature 486, 207‐214 (2012) doi:10.1038/nature11234C Huttenhower et al. Nature 486, 207‐214 (2012) doi:10.1038/nature112347