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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1997, p. 2338-2346 Copyright 0 1997, American Society for Microbiology 0099-2240/97/$04.00 + 0 Vol. 63. No. 6 A New Computational Method for Detection of Chimeric 16s rRNA Artifacts Generated by PCR Amplification from Mixed Bacterial Populations GEORGE A. KOMATSOULISlt AND MICHAEL S. WATERMANlT2* Departments of Mathematics' and Biological Sciences,2 University Southern California, Los Angeles, California 90089-1113 Received 16 January 1997/Accepted 26 March 1997 A new computational method (chimeric alignment) has been developed to detect chimeric 16s rRNA artifacts generated during PCR amplification from mixed bacterial populations. In contrast to other nearest-neighbor methods (e.g., CHECK-CHIMERA) that define sequence similarity by k-tuple matching, the chimeric align- ment method uses the score from dynamic programming alignments. Further, the chimeric alignments are displayed to the user to assist in sequence classification. The distribution of improvement scores for 500 authentic, nonchimeric sequences and 300 artificial chimeras (constructed from authentic sequences) was used to study the sensitivity and accuracy of both chimeric alignment and CHECK-CHIMERA. At a constant rate of authentic sequence misclassification (5%), chimeric alignment incorrectly classified 13% of the artificial chimeras versus 14% for CHECK-CHIMERA. Interestingly, only 1% of nonchimeras and 10% of chimeras were misclassified by both programs, suggesting that optimum performance is obtained by using the two methods to assign sequences to three classes: high-probability nonchimeras, high-probability chimeras, and sequences that need further study by other means. This study suggests that k-tuple-based matching methods are more sensitive than alignment-based methods when there is significant parental sequence similarity, while the opposite becomes true as the sequences become more distantly related. The software and a World Wide Web-based server are available at The use of 16s rRNA in the classification of bacterial species has been well established, and its effect on biology has been profound. It was 16s rRNA data that provided convincing evidence that chloroplasts and mitochondria most likely arose from free-living bacteria and that prokaryotic organisms rep- resented not one line of evolutionary descent but two, Bacteria and Archaea (previously eubacteria and archaebacteria), that diverged from each other and from the Eucaiya at approxi- mately the same time (5,26). The Ribosomal Database Project (RDP) (13) at the University of Illinois at Urbana-Champaign maintains an extensive, publicly accessible, database of 16s rRNA sequences with the long-term goal of developing com- plete phylogenies of all bacterial, archaebacterial, mitochon- drial, and chloroplast species.
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