title: Non-target sites with single nucleotide insertions or deletions are frequently found in 16S rRNA sequences and can lead to false positives in fluorescence in situ hybridization (FISH).
abstract: Fluorescence in situ hybridization (FISH) has impacted profoundly on our knowledge of the in situ ecophysiology and biodiversity of bacteria in natural communities. However, it has many technical challenges including the possibility of false positives from the binding of probes to non-target rRNA sequences. We show here that probe target sites containing single-base insertions or deletions can lead to false FISH positives, the result of hybridization with a bulge around the missing base. Experimental and in silico data suggest this situation occurs at a surprisingly high frequency. The existence of such sites is not currently considered during most FISH probe design processes. We describe software to identify potential non-target sites resulting from single-base insertions or deletions in rRNA sequences. This software also provides an estimate of the FISH probe hybridization efficiency to these sites.
authors.affiliation: Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3552, Australia.
authors.forename: Simon J
authors.initials: SJ
authors.lastname: McIlroy
authors.affiliation: Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3552, Australia.
authors.forename: Daniel
authors.initials: D
authors.lastname: Tillett
authors.affiliation: Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3552, Australia.
authors.forename: Steve
authors.initials: S
authors.lastname: Petrovski
authors.affiliation: Biotechnology Research Centre, La Trobe University, Bendigo, Victoria 3552, Australia.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Seviour
mesh_terms: D001419:Bacteria / Q000235:genetics; D001483:Base Sequence; D004269:DNA, Bacterial / Q000235:genetics; D054643:INDEL Mutation; D017404:In Situ Hybridization, Fluorescence; D015345:Oligonucleotide Probes / Q000737:chemistry*; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012984:Software
chemical_list: D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D012336:RNA, Ribosomal, 16S