Somatic DNA mutation load is a true molecular endpoint of an interaction between DNA damage and the DNA repair machinery and is the only direct indicator of a loss in genome sequence integrity. DNA mutations cause cancer and have also been implicated in other pathologies. For this reason, attempts have been made to develop assays for the quantitative analysis of various types of mutations in cells and tissues. This is important in genetic toxicology, in which the potential genetic risk associated with human exposure to the various damaging agents is evaluated. With the advent of next-generation sequencing somatic mutations can be quantitatively assessed, but only in clonal lineages, such as tumors, in which most cells share a substantial number of mutations. In normal tissues somatic mutations are of very low abundance and cannot be distinguished from sequencing errors. Utilization of single cell sequencing is the most sophisticated solution for this problem, but single cell-based approach is labor intensive and expensive which significantly limits its application for high throughput screening studies, a routine task in the field of genetic toxicology. An alternative approach, Duplex-seq, is utilizing bulk DNA as an input material and is based on comparative analysis of separately sequenced complementary DNA strands composing each DNA duplex. While technically less challenging than single cell-based technique, this method is still prohibitively costly due to unbalanced representation DNA strands resulting in very low rate of sequencing data utilization. Here we introduce Linked Strands Duplex-Seq (LSDS) assay which is designed to overcome this critical limitation of Duplex-seq. To accomplish that we designed a new sequencing library preparation procedure that ensures amplification of complementary DNA strands at equal rates. This will result in a completely balanced sequencing library and we expect to achieve at least a 10-fold increase in sequencing data utilization efficiency as compared to the original Duplex-seq approach. If successful, this method will fill the gap in genetic toxicology methodology and will provide a practical tool for accurate cost-effective genome-wide assessment of somatic mutational load in a high throughput manner.
Accurate identification of somatic mutations is important in genetic toxicology, in which the potential genetic risk associated with human exposure to the various damaging agents is evaluated. Here we propose to develop and optimize a novel assay for quantitative detection of somatic mutations in normal human cells and tissues using next generation sequencing. This method will fill the gap in genetic toxicology methodology and will provide a practical tool for accurate cost-effective genome-wide assessment of somatic mutational load in a high throughput manner.
