Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain.
Targeted enrichment and high-resolution digital profiling of mitochondrial DNA deletions in human brain.
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DOI：
10.1111/acel.12146
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发表时间：
2014-02
期刊：
影响因子：
7.8
通讯作者：
Bielas JH
中科院分区：
文献类型：
--
作者：
Taylor SD;Ericson NG;Burton JN;Prolla TA;Silber JR;Shendure J;Bielas JH
主要是由于无法准确地量化和表征从头缺失事件，在衰老和神经肌肉疾病的mtDNA缺失的致病性扩展的基础机制仍然知之甚少。在这里，我们概述并验证了一种名为“数字缺失检测”（3D）的新工具，该工具允许对频率低至1 × 10−8的罕见缺失进行高分辨率分析。3D是一个三步过程，包括对缺失分子的靶向富集，通过液滴数字PCR将基因组单分子分配到数千个液滴中进行直接定量，以及使用大规模平行测序进行断点表征。使用3D技术，我们询问了超过80亿个线粒体基因组，以分析人脑组织中mtDNA缺失的年龄相关动态。我们表明，总的删除负荷随年龄的增加，而独特的缺失的总数和多样性保持不变。我们的数据支持了这一假设，即既存突变的扩展是导致年龄相关的mtDNA缺失积累的主要因素。
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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