Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain

Authors

  • Sean D. Taylor,

    1. Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
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  • Nolan G. Ericson,

    1. Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
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  • Joshua N. Burton,

    1. Department of Genome Sciences, University of Washington, Seattle, WA, USA
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  • Tomas A. Prolla,

    1. Department of Medical Genetics, University of Wisconsin-Madison, Madison, WI, USA
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  • John R. Silber,

    1. Neurological Surgery, University of Washington Medical Center, Seattle, WA, USA
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  • Jay Shendure,

    1. Department of Genome Sciences, University of Washington, Seattle, WA, USA
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  • Jason H. Bielas

    Corresponding author
    1. Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
    2. Human Biology Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
    3. Department of Pathology, University of Washington Medical Center, Seattle, WA, USA
    • Correspondence

      Jason H. Bielas, Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, PO Box 19024, Mail Stop M5-A864, Seattle, WA 98109-1024, USA. Tel.: 206-667-3170; fax: 206-667-2537;

      e-mail: jbielas@fhcrc.org

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Summary

Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mtDNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed ‘Digital Deletion Detection’ (3D) that allows for high-resolution analysis of rare deletions occurring at frequencies as low as 1 × 10−8. 3D is a three-step process that includes targeted enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into thousands of droplets for direct quantification via droplet digital PCR, and breakpoint characterization using massively parallel sequencing. Using 3D, we interrogated over 8 billion mitochondrial genomes to analyze the age-related dynamics of mtDNA deletions in human brain tissue. We demonstrate that the total deletion load increases with age, while the total number and diversity of unique deletions remain constant. Our data provide support for the hypothesis that expansion of pre-existing mutations is the primary factor contributing to age-related accumulation of mtDNA deletions.

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