PMID- 24928876 OWN - NLM STAT- MEDLINE DCOM- 20150806 LR - 20211021 IS - 1098-5336 (Electronic) IS - 0099-2240 (Print) IS - 0099-2240 (Linking) VI - 80 IP - 16 DP - 2014 Aug TI - Automated design of probes for rRNA-targeted fluorescence in situ hybridization reveals the advantages of using dual probes for accurate identification. PG - 5124-33 LID - 10.1128/AEM.01685-14 [doi] AB - Fluorescence in situ hybridization (FISH) is a common technique for identifying cells in their natural environment and is often used to complement next-generation sequencing approaches as an integral part of the full-cycle rRNA approach. A major challenge in FISH is the design of oligonucleotide probes with high sensitivity and specificity to their target group. The rapidly expanding number of rRNA sequences has increased awareness of the number of potential nontargets for every FISH probe, making the design of new FISH probes challenging using traditional methods. In this study, we conducted a systematic analysis of published probes that revealed that many have insufficient coverage or specificity for their intended target group. Therefore, we developed an improved thermodynamic model of FISH that can be applied at any taxonomic level, used the model to systematically design probes for all recognized genera of bacteria and archaea, and identified potential cross-hybridizations for the selected probes. This analysis resulted in high-specificity probes for 35.6% of the genera when a single probe was used in the absence of competitor probes and for 60.9% when up to two competitor probes were used. Requiring the hybridization of two independent probes for positive identification further increased specificity. In this case, we could design highly specific probe sets for up to 68.5% of the genera without the use of competitor probes and 87.7% when up to two competitor probes were used. The probes designed in this study, as well as tools for designing new probes, are available online (http://DECIPHER.cee.wisc.edu). CI - Copyright (c) 2014, American Society for Microbiology. All Rights Reserved. FAU - Wright, Erik S AU - Wright ES AD - Department of Bacteriology, University of Wisconsin-Madison, Madison, Wisconsin, USA Systems Biology Theme, Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, Wisconsin, USA eswright@wisc.edu. FAU - Yilmaz, L Safak AU - Yilmaz LS AD - Program in Systems Biology, University of Massachusetts Medical School, Worcester, Massachusetts, USA. FAU - Corcoran, Andrew M AU - Corcoran AM AD - Department of Civil and Environmental Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA. FAU - Okten, Hatice E AU - Okten HE AD - Department of Environmental Engineering, Bahcesehir University, Istanbul, Turkey. FAU - Noguera, Daniel R AU - Noguera DR AD - Department of Civil and Environmental Engineering, University of Wisconsin-Madison, Madison, Wisconsin, USA. LA - eng PT - Evaluation Study PT - Journal Article PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20140613 PL - United States TA - Appl Environ Microbiol JT - Applied and environmental microbiology JID - 7605801 RN - 0 (DNA Primers) RN - 0 (DNA Probes) RN - 0 (DNA, Bacterial) RN - 0 (RNA, Ribosomal) SB - IM MH - Automation MH - Bacteria/classification/*genetics/isolation & purification MH - *Computers, Molecular MH - DNA Primers/chemistry/genetics MH - DNA Probes/*chemistry/genetics MH - DNA, Bacterial/*genetics MH - In Situ Hybridization, Fluorescence/instrumentation/methods MH - RNA, Ribosomal/*genetics PMC - PMC4135741 EDAT- 2014/06/15 06:00 MHDA- 2015/08/08 06:00 PMCR- 2015/02/01 CRDT- 2014/06/15 06:00 PHST- 2014/06/15 06:00 [entrez] PHST- 2014/06/15 06:00 [pubmed] PHST- 2015/08/08 06:00 [medline] PHST- 2015/02/01 00:00 [pmc-release] AID - AEM.01685-14 [pii] AID - 01685-14 [pii] AID - 10.1128/AEM.01685-14 [doi] PST - ppublish SO - Appl Environ Microbiol. 2014 Aug;80(16):5124-33. doi: 10.1128/AEM.01685-14. Epub 2014 Jun 13.