Development of an Efficient High Throughput Technique for the Identification of High-Impact Non-Coding Somatic Variants Across Multiple Tissue Types

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Project summary Somatic mutations accumulate in normal tissues and are increasingly recognized as a crucial determinant of disease risk, especially in age-related conditions and cancer. Somatic mutations show enrichment in portions of the noncoding genome that show “open” chromatin structure, such as active promoter and enhancer elements, because open chromatin is more vulnerable to mutagens. Furthermore, transcription factors binding appears to obstruct DNA repair, increasing the likelihood of forming fixed, double- stranded mutations. The channeling effects of these mechanisms result in a concentration of somatic mutations in restricted, yet critical, regions of the genome. Somatic mutations with an increased likelihood of causing diseases frequently arise at recurrent genomic sites, and often even recurrent mutations at specific bases, allowing for the development of targeted methods with greater sensitivity, lower cost, and higher throughput to identify somatic mutations than traditional sequencing techniques. Present methods for identifying somatic mutations generally utilize deep (≥250X) whole genome sequencing (WGS) and tend to be expensive, create large datasets that are computationally challenging to analyze, and have limited ability to detect somatic variants with very low allele fractions. We propose a two phase approach to developing a new tool to address these shortcomings. In the first phase we will develop a method of detecting somatic mutations using ATAC-seq. ATAC-seq targets the open chromatin regions of the genome so is focused on regions with increased somatic mutations that have an increased likelihood of being biologically meaningful, only incorporates a fraction of the genome creating a more manageable dataset, and allows for deeper sequencing to increase the sensitivity of somatic mutation detection. This phase of the proposal includes three aims: modification of the ATAC-seq protocol to allow for detection of somatic mutations; development of analysis software to analyze the data; and testing of the protocol. In the second phase of the protocol, data obtained from phase one will be used to develop a panel sequencing protocol to further narrow the genomic regions looked at, reduce the cost of the analysis, and allow for extracted DNA to be directly analyzed (rather than the intact chromatin needed for ATAC-seq). This phase will also involve three aims: expansion of ATAC-seq analysis to determine the best regions to include on the sequencing panel; development of the sequencing panel; and testing of the panel on a range of individuals and tissue types. This project will provide rapid and inexpensive methods for the detection of potentially critical somatic mutations in any tissue type. At a research level, it will allow for the analysis of a large number of samples to provide critical information on biologically important somatic mutations and thus be an important tool that will help illuminate the spectrum of somatic mutation in the noncoding genome.