The Hallmarks of Aging: Assessing accumulation of DNA lesions with age using single cell DNA sequencing in GESTALT

National Institute On Aging

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Abstract

Single-cell DNA sequencing is a new technology that can profile genetic mutations at the single-cell level and has the potential to dissect genetic heterogeneity at the highest resolution. For the proposed project, we amplified DNA from the collected single cells, performed a quality control of the DNA after amplification and performed DNA sequencing on high quality amplified DNA from 3-4 cells for each GESTALT participant. So far, we have successfully collected single-cell sequencing data from 119 monocytes extracted from the 30 participants. We then implemented the pipeline of SCcaller (Dong et al., Nature Methods, 2017) to identify somatic mutations in the nuclear DNA in each single cell while adjusting for sequencing coverage and sensitivity of the variant caller. Our preliminary analysis shows that the numbers of nuclear somatic mutations vary among the 3-4 single cells from the same individual and this large variation could be due to the unevenness of the DNA amplification from the single-cell multiple displacement amplification (SCMDA) process. Currently, we are applying a newer and more accurate single-cell variant identification software Scan-SNV to our data set in order to obtain a consensus somatic mutation set. We are also studying the mutational signatures of the identified somatic mutations (i.e., the unique combinations of mutation types generated by different mutational processes) and the effect of age on the top mutational signatures. Besides studying the somatic mutations in the nuclear genome, we are also testing whether the data can be used to study variation in the mitochondrial genome. We applied programs, designed specifically for analyzing mitochondrial genome, to sequencing data from each single cell and from merged single cells of the same individual. We were able to show for the first time that single-cell DNA sequencing can be used to identify mitochondrial DNA (mtDNA) variants both homoplasmies (variants affecting all of the mtDNA copies within a cell) and heteroplasmies (variants with more than one allele at a DNA site). We observed that single-cell sequencing identifies vast majority of homoplasmies observed in bulk DNA (DNA extracted from buffy coat), indicating the sharing of almost all homoplasmies between the buffy coat and monocytes. Furthermore, single-cell sequencing captures additional heteroplasmies that were not observed in bulk DNA, indicating many heteroplasmies are acquired as somatic mutations in a cell-specific manner. In addition, heteroplasmies unique to single-cell DNA are more likely to be deleterious, further supporting that somatic mutations are often acquired by cells individually.