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

Aging Cell

Sean D. Taylor¹  Nolan G. Ericson¹  Joshua N. Burton⁴  Tomas A. Prolla²  John R. Silber³  Jay Shendure⁴ 

[1] Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA;Department of Medical Genetics, University of Wisconsin-Madison, Madison, WI, USA;Neurological Surgery, University of Washington Medical Center, Seattle, WA, USA;Department of Genome Sciences, University of Washington, Seattle, WA, USA

关键词: aging;    genome instability;    mitochondrial disease;    mitochondrial DNA;    next‐generation sequencing;    rare deletion detection;   

DOI  :  10.1111/acel.12146

来源: Wiley

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.