Publications

Researchers from Health Canada recently published a paper revealing a positive correlation between Duplex Sequencing (DS) technology and the current “gold-standard” transgenic rodent (TGR) assay to assess mutagenicity. The inter-laboratory validation study indicated consistent results across laboratories (Health Canada and TwinStrand Biosciences). The findings in this study show that DS yields novel insights into the mutagenic mode of action, which may help to overcome limitations of current mutagenicity assays for future regulatory decision making.

The organotypic human air-liquid-interface (ALI) airway tissue model has been used as an in vitro cell culture system for evaluating the toxicity of inhaled substances. ALI airway cultures are highly differentiated, which has made it challenging to evaluate genetic toxicology endpoints. In the current study, we assayed DNA damage with the high-throughput CometChip assay and quantified mutagenesis with Duplex Sequencing, an error-corrected next-generation sequencing method capable of detecting a single mutation per 107 base pairs. Fully differentiated human ALI airway cultures were treated from the basolateral side with 6.25 to 100 μg/mL ethyl methanesulfonate (EMS) over a period of 28 days. CometChip assays were conducted after 3 and 28 days of treatment, and Duplex Sequencing after 28 days of treatment. Treating the airway cultures with EMS resulted in time- and concentration-dependent increases in DNA damage and a concentration-dependent increase in mutant frequency. The mutations observed in the EMS-treated cultures were predominantly C → T transitions and exhibited a unique trinucleotide signature relative to the negative control. Measurement of physiological endpoints indicated that the EMS treatments had no effect on anti-p63-positive basal cell frequency, but produced concentration-responsive increases in cytotoxicity and perturbations in cell morphology, along with concentration-responsive decreases in culture viability, goblet cell and anti-Ki67-positive proliferating cell frequency, cilia beating frequency, and mucin secretion. The results indicate that a unified 28-day study can be used to measure several important safety endpoints in physiologically relevant human in vitro ALI airway cultures, including DNA damage, mutagenicity, and tissue-specific general toxicity.

Next-generation DNA sequencing promises to revolutionize clinical medicine and basic research. However, while this technology has the capacity to generate hundreds of billions of nucleotides of DNA sequence in a single experiment, the error rate of ∼1% results in hundreds of millions of sequencing mistakes. These scattered errors can be tolerated in some applications but become extremely problematic when “deep sequencing” genetically heterogeneous mixtures, such as tumors or mixed microbial populations. To overcome limitations in sequencing accuracy, we have developed a method termed Duplex Sequencing. This approach greatly reduces errors by independently tagging and sequencing each of the two strands of a DNA duplex. As the two strands are complementary, true mutations are found at the same position in both strands. In contrast, PCR or sequencing errors result in mutations in only one strand and can thus be discounted as technical error. We determine that Duplex Sequencing has a theoretical background error rate of less than one artifactual mutation per billion nucleotides sequenced. In addition, we establish that detection of mutations present in only one of the two strands of duplex DNA can be used to identify sites of DNA damage.  We apply the method to directly assess the frequency and pattern of random mutations in mitochondrial DNA from human cells.

Mutations have a profound effect on human health, particularly through an increased risk of carcinogenesis and genetic disease. The strong correlation between mutagenesis and carcinogenesis has been a driving force behind genotoxicity research for more than 50 years. The stochastic and infrequent nature of mutagenesis makes it challenging to observe and to study. Indeed, decades have been spent developing increasingly sophisticated assays and methods to study these low-frequency genetic errors, in hopes of better predicting which chemicals may be carcinogens, understanding their mode of action, and informing guidelines to prevent undue human exposure. While effective, widely used genetic selection-based technologies have a number of limitations that have hampered major advancements in the field of genotoxicity. Emerging new tools, in the form of enhanced next-generation sequencing platforms and methods, are changing this paradigm. In this review, we discuss rapidly evolving sequencing tools and technologies, such as error-corrected sequencing and single cell analysis, which we anticipate will fundamentally reshape the field. In addition, we consider a variety emerging applications for these new technologies, including the detection of DNA adducts, inference of mutational processes based on genomic site and local sequence contexts, and evaluation of genome engineering fidelity, as well as other cutting-edge challenges for the next 50 years of environmental and molecular mutagenesis research.