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By benchmarking CS performance against sequences derived from a controlled DNA mixture of known organisms and a simulated chimera set, we provide insights into the factors that affect chimera formation such as sequence abundance, the extent of similarity between 16S genes, and PCR conditions.Chimeras were found to reproducibly form among independent amplifications and contributed to false perceptions of sample diversity and the false identification of novel taxa, with less-abundant species exhibiting chimera rates exceeding 70%.Adapting the 454 sequencing to analyze pilin Av demonstrates the utility of this technology to analyze any diversity generation system that uses recombination to develop biological diversity.

(the gonococcus [Gc]) is the sole causative agent of the sexually transmitted infection gonorrhea, with an estimated 800,000 new cases per year in the United States (1) and an estimated 106 million cases worldwide (2).

The high-sequence conservation of 16S genes among diverse bacteria allows for the phylogenetic analysis of organism diversity and the identification of new taxa. Despite efforts by the curators to remove low-quality sequences from survey data, it is likely that many of these reference sequences reflect sequencing artifacts rather than real biological diversity. Multiple factors including pairwise sequence identity between 16S r RNA genes, number of PCR cycles, and relative abundance of gene-specific PCR templates have been shown to influence chimera formation (Wang and Wang 1996, 1997; Thompson et al. Although chimera formation rates can be lowered experimentally, no method has been shown to eliminate these artifacts entirely. 2006a), used by the Green Genes 16S r RNA sequence collection (De Santis et al. The 16S-specific Bellerophon algorithm developed at Green Genes differs from the more general Bellerophon algorithm published earlier by Huber et al.

, the bacterium responsible for the sexually transmitted infection gonorrhea, achieves this in part by changing the sequence of the major subunit of the type IV pilus in a process termed pilin antigenic variation (Av). We have developed a 454 sequencing-based assay to analyze the frequency and characteristics of pilin Av that allows a more robust analysis of pilin Av than previous assays.

Perceived diversity, however, can be influenced by sample preparation, primer selection, and formation of chimeric 16S amplification products.

Chimeras are hybrid products between multiple parent sequences that can be falsely interpreted as novel organisms, thus inflating apparent diversity.

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