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Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons.

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Haas, Brian J. Gevers, Dirk Earl, Ashlee M. Feldgarden, Mike Ward, Doyle V. Giannoukos, Georgia Ciulla, Dawn Tabbaa, Diana Highlander, Sarah K. Sodergren, Erica Methé, Barbara DeSantis, Todd Z. Petrosino, Joseph F. Knight, Rob Birren, Bruce W.
Organization	Human Microbiome Consortium
Description	Country affiliation: United States Author Affiliation: Haas BJ ( Genome Sequencing and Analysis Program, The Broad Institute, Cambridge, Massachusetts 02142, USA.)
Abstract	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.
File Format	HTM / HTML
ISSN	10889051
e-ISSN	15495469
DOI	10.1101/gr.112730.110
Journal	Genome Research
Issue Number	3
Volume Number	21
Language	English
Publisher	Cold Spring Harbor Laboratory Press
Publisher Date	2011-03-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Discipline Genetics Discipline Genomics Artifacts Bacteria Genetics Rna, Ribosomal, 16s Classification Chimera Dna, Bacterial Dna, Ribosomal Genomics Molecular Sequence Data Nucleic Acid Amplification Techniques Polymerase Chain Reaction Rna, Bacterial Sequence Analysis, Dna Research Support, N.i.h., Extramural Research Support, Non-u.s. Gov't
Content Type	Text
Resource Type	Article
Subject	Genetics Genetics (clinical)

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Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons

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Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons.

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