Developing Methods for Enhanced Measurement of DNA Single-Strand Breaks and Somatic Variants

Abstract

Maintenance and repair of DNA are essential for proper cellular functioning and preventing the emergence of disease states. As cells divide, mutations accumulate in the genome which contributes to aging phenotypes and can result in genetic diseases such as cancer. The rate at which a cell develops mutations can be accelerated through exposure to genotoxic agents that introduce lesions which, if left unrepaired, prevent accurate replication of the genome. As such, it is crucial to understand the ways in which DNA becomes damaged, how cells respond to various types of damage, and how this damage contributes to mutagenesis and the development of genetic disease. These fields of study have been greatly advanced by improvements in DNA sequencing technologies, and here we present two sequencing-based methods that aim to enable deeper study of DNA damage, repair, and mutagenesis. First, we demonstrate DENT-seq, a method that identifies single-strand breaks with single-nucleotide resolution. Single-strand breaks are the most common form of DNA damage, occurring at rates of ~10,000 per cell per day, but have to date been understudied due to lack of an unbiased, high-resolution method for their detection. Second, we improve upon lineage sequencing, a previously reported method that uniquely measures somatic single nucleotide variants in dividing cells to achieve high specificity/sensitivity as well as the ability to temporally resolve variants and to relate sequenced genotypes to optically observed cellular phenotypes. Despite the high-quality data and unique capabilities offered by this method, it has so far been underused due to a need for complex, microfluidic-based cell collection. We demonstrate novel protocols for performing lineage sequencing that enable easy adoption of the method without the need for highly specialized equipment or expertise. In addition, we expand the repertoire of mutations measurable with the technique to include indels and variants that arise specifically in response to a genotoxic treatment. The methods we show can be applied to reveal novel findings regarding the causes and consequences of DNA damage and mutagenesis that underly numerous genetic diseases.Maintenance and repair of DNA are essential for proper cellular functioning and preventing the emergence of disease states. As cells divide, mutations accumulate in the genome which contributes to aging phenotypes and can result in genetic diseases such as cancer. The rate at which a cell develops mutations can be accelerated through exposure to genotoxic agents that introduce lesions which, if left unrepaired, prevent accurate replication of the genome. As such, it is crucial to understand the ways in which DNA becomes damaged, how cells respond to various types of damage, and how this damage contributes to mutagenesis and the development of genetic disease. These fields of study have been greatly advanced by improvements in DNA sequencing technologies, and here we present two sequencing-based methods that aim to enable deeper study of DNA damage, repair, and mutagenesis. First, we demonstrate DENT-seq, a method that identifies single-strand breaks with single-nucleotide resolution. Single-strand breaks are the most common form of DNA damage, occurring at rates of ~10,000 per cell per day, but have to date been understudied due to lack of an unbiased, high-resolution method for their detection. Second, we improve upon lineage sequencing, a previously reported method that uniquely measures somatic single nucleotide variants in dividing cells to achieve high specificity/sensitivity as well as the ability to temporally resolve variants and to relate sequenced genotypes to optically observed cellular phenotypes. Despite the high-quality data and unique capabilities offered by this method, it has so far been underused due to a need for complex, microfluidic-based cell collection. We demonstrate novel protocols for performing lineage sequencing that enable easy adoption of the method without the need for highly specialized equipment or expertise. In addition, we expand the repertoire of mutations measurable with the technique to include indels and variants that arise specifically in response to a genotoxic treatment. The methods we show can be applied to reveal novel findings regarding the causes and consequences of DNA damage and mutagenesis that underly numerous genetic diseases.