title: Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons.
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.
authors.affiliation: Genome Sequencing and Analysis Program, The Broad Institute, Cambridge, Massachusetts 02142, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Haas
authors.forename: Dirk
authors.initials: D
authors.lastname: Gevers
authors.forename: Ashlee M
authors.initials: AM
authors.lastname: Earl
authors.forename: Mike
authors.initials: M
authors.lastname: Feldgarden
authors.forename: Doyle V
authors.initials: DV
authors.lastname: Ward
authors.forename: Georgia
authors.initials: G
authors.lastname: Giannoukos
authors.forename: Dawn
authors.initials: D
authors.lastname: Ciulla
authors.forename: Diana
authors.initials: D
authors.lastname: Tabbaa
authors.forename: Sarah K
authors.initials: SK
authors.lastname: Highlander
authors.forename: Erica
authors.initials: E
authors.lastname: Sodergren
authors.forename: Barbara
authors.initials: B
authors.lastname: Methé
authors.forename: Todd Z
authors.initials: TZ
authors.lastname: DeSantis
authors.forename: Joseph F
authors.initials: JF
authors.lastname: Petrosino
authors.forename: Rob
authors.initials: R
authors.lastname: Knight
authors.forename: Bruce W
authors.initials: BW
authors.lastname: Birren
mesh_terms: D016477:Artifacts; D001419:Bacteria / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D002678:Chimera / Q000235:genetics; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D023281:Genomics; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods; D012329:RNA, Bacterial / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000032:analysis*; D017422:Sequence Analysis, DNA / Q000379:methods
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S