Sources of PCR-induced distortions in high-throughput sequencing data sets
被引:172
作者:
Kebschull, Justus M.
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机构:
Cold Spring Harbor Lab, Watson Sch Biol Sci, Cold Spring Harbor, NY 11724 USA
Cold Spring Harbor Lab, Cold Spring Harbor, NY 11724 USACold Spring Harbor Lab, Watson Sch Biol Sci, Cold Spring Harbor, NY 11724 USA
Kebschull, Justus M.
[1
,2
]
Zador, Anthony M.
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机构:
Cold Spring Harbor Lab, Cold Spring Harbor, NY 11724 USACold Spring Harbor Lab, Watson Sch Biol Sci, Cold Spring Harbor, NY 11724 USA
Zador, Anthony M.
[2
]
机构:
[1] Cold Spring Harbor Lab, Watson Sch Biol Sci, Cold Spring Harbor, NY 11724 USA
[2] Cold Spring Harbor Lab, Cold Spring Harbor, NY 11724 USA
PCR permits the exponential and sequence-specific amplification of DNA, even from minute starting quantities. PCR is a fundamental step in preparing DNA samples for high-throughput sequencing. However, there are errors associated with PCR-mediated amplification. Here we examine the effects of four important sources of error-bias, stochasticity, template switches and polymerase errors-on sequence representation in low-input next-generation sequencing libraries. We designed a pool of diverse PCR amplicons with a defined structure, and then used Illumina sequencing to search for signatures of each process. We further developed quantitative models for each process, and compared predictions of these models to our experimental data. We find that PCR stochasticity is the major force skewing sequence representation after amplification of a pool of unique DNA amplicons. Polymerase errors become very common in later cycles of PCR but have little impact on the overall sequence distribution as they are confined to small copy numbers. PCR template switches are rare and confined to low copy numbers. Our results provide a theoretical basis for removing distortions from high-throughput sequencing data. In addition, our findings on PCR stochasticity will have particular relevance to quantification of results from single cell sequencing, in which sequences are represented by only one or a few molecules.
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Haas, Brian J.
Gevers, Dirk
论文数: 0引用数: 0
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Gevers, Dirk
Earl, Ashlee M.
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h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Earl, Ashlee M.
Feldgarden, Mike
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Feldgarden, Mike
Ward, Doyle V.
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Ward, Doyle V.
Giannoukos, Georgia
论文数: 0引用数: 0
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机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Giannoukos, Georgia
Ciulla, Dawn
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h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Ciulla, Dawn
Tabbaa, Diana
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Tabbaa, Diana
Highlander, Sarah K.
论文数: 0引用数: 0
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 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Highlander, Sarah K.
Sodergren, Erica
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h-index: 0
机构:
Washington Univ, Sch Med, Genome Ctr, St Louis, MO 63108 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Sodergren, Erica
Methe, Barbara
论文数: 0引用数: 0
h-index: 0
机构:
J Craig Venter Inst, Rockville, MD 20850 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Methe, Barbara
DeSantis, Todd Z.
论文数: 0引用数: 0
h-index: 0
机构:
Lawrence Berkeley Natl Lab, Div Earth Sci, Berkeley, CA 94720 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
DeSantis, Todd Z.
Petrosino, Joseph F.
论文数: 0引用数: 0
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 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Petrosino, Joseph F.
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 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Knight, Rob
Birren, Bruce W.
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Haas, Brian J.
Gevers, Dirk
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Gevers, Dirk
Earl, Ashlee M.
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Earl, Ashlee M.
Feldgarden, Mike
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Feldgarden, Mike
Ward, Doyle V.
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Ward, Doyle V.
Giannoukos, Georgia
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Giannoukos, Georgia
Ciulla, Dawn
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Ciulla, Dawn
Tabbaa, Diana
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Tabbaa, Diana
Highlander, Sarah K.
论文数: 0引用数: 0
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 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Highlander, Sarah K.
Sodergren, Erica
论文数: 0引用数: 0
h-index: 0
机构:
Washington Univ, Sch Med, Genome Ctr, St Louis, MO 63108 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Sodergren, Erica
Methe, Barbara
论文数: 0引用数: 0
h-index: 0
机构:
J Craig Venter Inst, Rockville, MD 20850 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Methe, Barbara
DeSantis, Todd Z.
论文数: 0引用数: 0
h-index: 0
机构:
Lawrence Berkeley Natl Lab, Div Earth Sci, Berkeley, CA 94720 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
DeSantis, Todd Z.
Petrosino, Joseph F.
论文数: 0引用数: 0
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 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Petrosino, Joseph F.
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 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA
Knight, Rob
Birren, Bruce W.
论文数: 0引用数: 0
h-index: 0
机构:
Broad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USABroad Inst, Genome Sequencing & Anal Program, Cambridge, MA 02142 USA