title: Targeted and highly multiplexed detection of microorganisms by employing an ensemble of molecular probes.
abstract: The vast majority of microscopic life on earth consists of microbes that do not grow in laboratory culture. To profile the microbial diversity in environmental and clinical samples, we have devised and employed molecular probe technology, which detects and identifies bacteria that do and do not grow in culture. The only requirement is a short sequence of contiguous bases (currently 60 bases) unique to the genome of the organism of interest. The procedure is relatively fast, inexpensive, customizable, robust, and culture independent and uses commercially available reagents and instruments. In this communication, we report improving the specificity of the molecular probes substantially and increasing the complexity of the molecular probe set by over an order of magnitude (>1,200 probes) and introduce a new final readout method based upon Illumina sequencing. In addition, we employed molecular probes to identify the bacteria from vaginal swabs and demonstrate how a deliberate selection of molecular probes can identify less abundant bacteria even in the presence of much more abundant species.
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA.
authors.forename: Weihong
authors.initials: W
authors.lastname: Xu
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA.
authors.forename: Sujatha
authors.initials: S
authors.lastname: Krishnakumar
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA.
authors.forename: Molly
authors.initials: M
authors.lastname: Miranda
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Jensen
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA.
authors.forename: Marilyn
authors.initials: M
authors.lastname: Fukushima
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA.
authors.forename: Curtis
authors.initials: C
authors.lastname: Palm
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA.
authors.forename: Eula
authors.initials: E
authors.lastname: Fung
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA Department of Genetics, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Davis
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA.
authors.forename: Robert P
authors.initials: RP
authors.lastname: St Onge
authors.affiliation: Stanford Genome Technology Center, Palo Alto, California, USA Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA rhyman@stanford.edu.
authors.forename: Richard W
authors.initials: RW
authors.lastname: Hyman
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D001431:Bacteriological Techniques / Q000379:methods; D004269:DNA, Bacterial / Q000235:genetics; D015335:Molecular Probes / Q000737:chemistry*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D009841:Oligonucleotides / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D015335:Molecular Probes; D009841:Oligonucleotides