共 37 条
Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons
被引:2899
作者:

Haas, Brian J.
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Gevers, Dirk
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Earl, Ashlee M.
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Feldgarden, Mike
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Ward, Doyle V.
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Giannoukos, Georgia
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Ciulla, Dawn
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Tabbaa, Diana
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Highlander, Sarah K.
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机构:
Baylor Coll Med, Human Genome Sequencing Ctr, Houston, TX 77030 USA
Baylor Coll Med, Dept Mol Virol & Microbiol, Houston, TX 77030 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Sodergren, Erica
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机构:
Washington Univ, Sch Med, Genome Ctr, St Louis, MO 63108 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Methe, Barbara
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机构:
J Craig Venter Inst, Rockville, MD 20850 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

DeSantis, Todd Z.
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机构:
Lawrence Berkeley Natl Lab, Div Earth Sci, Berkeley, CA 94720 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Petrosino, Joseph F.
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h-index: 0
机构:
Baylor Coll Med, Human Genome Sequencing Ctr, Houston, TX 77030 USA
Baylor Coll Med, Dept Mol Virol & Microbiol, Houston, TX 77030 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Knight, Rob
论文数: 0 引用数: 0
h-index: 0
机构:
Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA
Univ Colorado, Howard Hughes Med Inst, Boulder, CO 80309 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA

Birren, Bruce W.
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
机构:
[1] Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
[2] Baylor Coll Med, Human Genome Sequencing Ctr, Houston, TX 77030 USA
[3] Baylor Coll Med, Dept Mol Virol & Microbiol, Houston, TX 77030 USA
[4] Washington Univ, Sch Med, Genome Ctr, St Louis, MO 63108 USA
[5] J Craig Venter Inst, Rockville, MD 20850 USA
[6] Lawrence Berkeley Natl Lab, Div Earth Sci, Berkeley, CA 94720 USA
[7] Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA
[8] Univ Colorado, Howard Hughes Med Inst, Boulder, CO 80309 USA
关键词:
MICROBIAL DIVERSITY;
RARE BIOSPHERE;
INTRAGENOMIC HETEROGENEITY;
INTERGENOMIC RECOMBINATION;
GENES;
CONSEQUENCE;
LIBRARIES;
COAMPLIFICATION;
AMPLIFICATION;
ALIGNMENTS;
D O I:
10.1101/gr.112730.110
中图分类号:
Q5 [生物化学];
Q7 [分子生物学];
学科分类号:
071010 ;
081704 ;
摘要:
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.
引用
收藏
页码:494 / 504
页数:11
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机构: Lawrence Berkeley Natl Lab, Ctr Environm Biotechnol, Berkeley, CA 94720 USA

Hugenholtz, P.
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Larsen, N.
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Rojas, M.
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Brodie, E. L.
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机构: Lawrence Berkeley Natl Lab, Ctr Environm Biotechnol, Berkeley, CA 94720 USA

Keller, K.
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Huber, T.
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Dalevi, D.
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Hu, P.
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Andersen, G. L.
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[10]
Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex
[J].
Hamady, Micah
;
Walker, Jeffrey J.
;
Harris, J. Kirk
;
Gold, Nicholas J.
;
Knight, Rob
.
NATURE METHODS,
2008, 5 (03)
:235-237

Hamady, Micah
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机构:
Univ Colorado, Dept Comp Sci, Boulder, CO 80309 USA Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA

Walker, Jeffrey J.
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机构:
Univ Colorado, Dept Mol Cellular & Dev Biol, Boulder, CO 80309 USA Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA

Harris, J. Kirk
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机构:
Univ Colorado, Dept Pediat, Aurora, CO 80045 USA
Childrens Hosp, Ctr Hlth Sci, Aurora, CO 80045 USA Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA

Gold, Nicholas J.
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Univ Colorado, Dept Mol Cellular & Dev Biol, Boulder, CO 80309 USA Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA

Knight, Rob
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机构:
Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA Univ Colorado, Dept Chem & Biochem, Boulder, CO 80309 USA