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Applied and Environmental Microbiology, September 2006, p. 5734-5741, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00556-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

New Screening Software Shows that Most Recent Large 16S rRNA Gene Clone Libraries Contain Chimeras{dagger}

Kevin E. Ashelford,1* Nadia A. Chuzhanova,2 John C. Fry,1 Antonia J. Jones,3 and Andrew J. Weightman1

Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, PO Box 915, Cardiff CF10 3TL, United Kingdom,1 Biostatistics and Bioinformatics Unit and Institute of Medical Genetics, Cardiff School of Medicine, Cardiff University, Heath Park, Cardiff CF14 4XN, United Kingdom,2 Cardiff School of Computer Science, Cardiff University, Queen's Buildings, 5 The Parade, Roath, Cardiff CF24 3AA, United Kingdom3

Received 8 March 2006/ Accepted 9 June 2006

A new computer program, called Mallard, is presented for screening entire 16S rRNA gene libraries of up to 1,000 sequences for chimeras and other artifacts. Written in the Java computer language and capable of running on all major operating systems, the program provides a novel graphical approach for visualizing phylogenetic relationships among 16S rRNA gene sequences. To illustrate its use, we analyzed most of the large libraries of cloned bacterial 16S rRNA gene sequences submitted to the public repository during 2005. Defining a large library as one containing 100 or more sequences of 1,200 bases or greater, we screened 25 of the 28 libraries and found that all but three contained substantial anomalies. Overall, 543 anomalous sequences were found. The average anomaly content per clone library was 9.0%, 4% higher than that previously estimated for the public repository overall. In addition, 90.8% of anomalies had characteristic chimeric patterns, a rise of 25.4% over that found previously. One library alone was found to contain 54 chimeras, representing 45.8% of its content. These figures far exceed previous estimates of artifacts within public repositories and further highlight the urgent need for all researchers to adequately screen their libraries prior to submission. Mallard is freely available from our website at http://www.cardiff.ac.uk/biosi/research/biosoft/.

* Corresponding author. Mailing address: Cardiff School of Biosciences, Cardiff University, Main Building, Park Place, P.O. Box 915, Cardiff CF10 3TL, United Kingdom. Phone: 44 (0)29 20 876002. Fax: 44 (0)29 20 874305. E-mail: ashelford{at}cardiff.ac.uk.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, September 2006, p. 5734-5741, Vol. 72, No. 9
0099-2240/06/$08.00+0     doi:10.1128/AEM.00556-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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