RT Journal
A1 Haas, Brian J.
A1 Gevers, Dirk
A1 Earl, Ashlee M.
A1 Feldgarden, Mike
A1 Ward, Doyle V.
A1 Giannoukos, Georgia
A1 Ciulla, Dawn
A1 Tabbaa, Diana
A1 Highlander, Sarah K.
A1 Sodergren, Erica
A1 Methé, Barbara
A1 DeSantis, Todd Z.
A1 The Human Microbiome Consortium
A1 Petrosino, Joseph F.
A1 Knight, Rob
A1 Birren, Bruce W.
T1 Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons
JF Genome Research 
JO Genome Research 
YR 2011 
FD March 01 
VO 21 
IS 3 
SP 494 
OP 504 
DO 10.1101/gr.112730.110 
UL http://genome.cshlp.org/content/21/3/494.abstract 
AB Bacterial diversity among environmental samples is commonly assessed with PCR-amplified 16S rRNA gene (16S) sequences. 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. We developed a new chimera detection tool called Chimera Slayer (CS). CS detects chimeras with greater sensitivity than previous methods, performs well on short sequences such as those produced by the 454 Life Sciences (Roche) Genome Sequencer, and can scale to large data sets. 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%. Shotgun metagenomic sequences of our mock community appear to be devoid of 16S chimeras, supporting a role for shotgun metagenomics in validating novel organisms discovered in targeted sequence surveys.