Open AccessTargeted enrichment and high‐resolution digital profiling of mitochondrial DNA deletions in human brain
Author(s) -
Taylor Sean D.,
Ericson Nolan G.,
Burton Joshua N.,
Prolla Tomas A.,
Silber John R.,
Shendure Jay,
Bielas Jason H.
Publication year - 2014
Publication title -
aging cell
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.103
H-Index - 140
eISSN - 1474-9726
pISSN - 1474-9718
DOI - 10.1111/acel.12146
Subject(s) - biology , breakpoint , mitochondrial dna , digital polymerase chain reaction , genetics , computational biology , genome , dna , gene , polymerase chain reaction , chromosomal translocation
Summary Due largely to the inability to accurately quantify and characterize de novo deletion events, the mechanisms underpinning the pathogenic expansion of mt DNA deletions in aging and neuromuscular disorders remain poorly understood. Here, we outline and validate a new tool termed ‘ D igital D eletion D etection’ (3 D ) that allows for high‐resolution analysis of rare deletions occurring at frequencies as low as 1 × 10 −8 . 3 D 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 3 D , we interrogated over 8 billion mitochondrial genomes to analyze the age‐related dynamics of mt DNA 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 mt DNA deletions.