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)
Article first published online: 22 JUL 2010
© 2010 Society for Applied Microbiology and Blackwell Publishing Ltd
Volume 13, Issue 1, pages 33–47, January 2011
How to Cite
McIlroy, S. J., Tillett, D., Petrovski, S. and Seviour, R. J. (2011), 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). Environmental Microbiology, 13: 33–47. doi: 10.1111/j.1462-2920.2010.02306.x
- Issue published online: 4 JAN 2011
- Article first published online: 22 JUL 2010
- Received 5 August, 2009; accepted 9 June, 2010.
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.