Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain
Article first published online: 11 SEP 2013
© 2013 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 13, Issue 1, pages 29–38, February 2014
How to Cite
Taylor, S. D., Ericson, N. G., Burton, J. N., Prolla, T. A., Silber, J. R., Shendure, J. and Bielas, J. H. (2014), Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain. Aging Cell, 13: 29–38. doi: 10.1111/acel.12146
- Issue published online: 16 JAN 2014
- Article first published online: 11 SEP 2013
- Accepted manuscript online: 2 AUG 2013 10:56AM EST
- Manuscript Accepted: 21 JUL 2013
- CDMRP/U.S. Department of Defense. Grant Numbers: W81XWH-10-1-0563, AG-NS-0577-09
- Ellison Medical Foundation. Grant Number: RO1 ES019319
- National Institute of Environmental Health Sciences of the National Institutes of Health. Grant Number: F32ES021703
- UW Genome Sciences
- genome instability;
- mitochondrial disease;
- mitochondrial DNA;
- next-generation sequencing;
- rare deletion detection
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.