MADDD-seq, a novel massively parallel sequencing tool for simultaneous detection of DNA damage and mutations

Abstract

Our genome is exposed to a wide variety of DNA-damaging agents. If left unrepaired, this damage can be fixed into mutations that promote carcinogenesis and the development of genetically inherited diseases. As a result, it is crucial that we can detect DNA damage and mutations with exquisite sensitivity. Here, we describe a modified version of double barcoding sequencing technology termed Mutation And DNA Damage Detection-seq (MADDD-seq) that can detect DNA damage and mutations simultaneously, with a single assay. To demonstrate the utility of MADDD-seq as a multifunctional detection tool, we treated yeast cells with a DNA-damaging agent and tracked the presence of DNA damage and mutations over a 24-hour timespan. These experiments allowed us to identify thousands of adducts and mutations in a single sequencing run and expose the kinetics of DNA repair and mutagenesis in remarkable detail.

The cycad genotoxin methylazoxymethanol, linked to Guam ALS/PDC, induces transcriptional mutagenesis

“…Such comparisons would not be feasible using bulk RNA-sequencing, because reads in bulk RNA-seq data may correspond to distinct DNA lesions in different cells. This scRNA-seq strategy has recently been used in another study [ 21 ], in which arrested yeast cells and quiescent mouse neural stem cells (NSCs) were exposed to N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG), a chemical compound that is routinely used to induce O 6 -mG DNA lesions and RNA synthesis errors [ 16 , 22 ], and subsequently used as input material for scRNA-seq. It was found that certain transcription errors do indeed occur repetitively as a consequence of MNNG exposure.…”