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Targeted genome fragmentation with CRISPR/Cas9 improves hybridization capture, reduces PCR bias, and enables efficient high-accuracy sequencing of small targets

Daniela Nachmanson, Shenyi Lian, Elizabeth K. Schmidt, Michael J. Hipp, Kathryn T. Baker, Yuezheng Zhang, Maria Tretiakova, Kaitlyn Loubet-Senear, Brendan F. Kohrn, Jesse J. Salk, Scott R. Kennedy, Rosa Ana Risques
doi: https://doi.org/10.1101/207027
Daniela Nachmanson
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Shenyi Lian
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Elizabeth K. Schmidt
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Michael J. Hipp
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Kathryn T. Baker
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Yuezheng Zhang
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Maria Tretiakova
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Kaitlyn Loubet-Senear
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Brendan F. Kohrn
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Jesse J. Salk
Department of Medicine, Division of Hematology and Oncology, University of Washington, Seattle, WA 98195, USA.
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Scott R. Kennedy
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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Rosa Ana Risques
Department of Pathology, University of Washington, Seattle, WA 98195, USA.
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ABSTRACT

Current next-generation sequencing techniques suffer from inefficient target enrichment and frequent errors. To address these issues, we have developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion. By designing all fragments to similar lengths, regions of interest can be size-selected prior to library preparation, increasing hybridization capture efficiency. Additionally, homogenous length fragments reduce PCR bias and maximize read usability. We combine this novel target enrichment approach with ultra-accurate Duplex Sequencing. The result, termed CRISPR-DS, is a robust targeted sequencing technique that overcomes the inherent challenges of small target enrichment and enables the detection of ultra-low frequency mutations with small DNA inputs.

  • ABBREVIATIONS

    DS
    Duplex Sequencing
    DCS
    Double-stranded consensus sequence
    SSCS
    Single-stranded consensus sequence
    gRNA
    Guide RNA
    crRNA
    CRISPR RNA
    tracrRNA
    Trans-activating crRNA
    NGS
    Next-generation Sequencing
    ng
    Nanogram
    bp
    Basepair
    ssDNA
    Single-stranded DNA
    dsDNA
    Double-stranded DNA
    DIN
    DNA integrity number
  • Copyright 
    The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license.
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    Posted January 12, 2018.
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    Targeted genome fragmentation with CRISPR/Cas9 improves hybridization capture, reduces PCR bias, and enables efficient high-accuracy sequencing of small targets
    Daniela Nachmanson, Shenyi Lian, Elizabeth K. Schmidt, Michael J. Hipp, Kathryn T. Baker, Yuezheng Zhang, Maria Tretiakova, Kaitlyn Loubet-Senear, Brendan F. Kohrn, Jesse J. Salk, Scott R. Kennedy, Rosa Ana Risques
    bioRxiv 207027; doi: https://doi.org/10.1101/207027
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    Targeted genome fragmentation with CRISPR/Cas9 improves hybridization capture, reduces PCR bias, and enables efficient high-accuracy sequencing of small targets
    Daniela Nachmanson, Shenyi Lian, Elizabeth K. Schmidt, Michael J. Hipp, Kathryn T. Baker, Yuezheng Zhang, Maria Tretiakova, Kaitlyn Loubet-Senear, Brendan F. Kohrn, Jesse J. Salk, Scott R. Kennedy, Rosa Ana Risques
    bioRxiv 207027; doi: https://doi.org/10.1101/207027

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