Development of a Genotoxicity Assay using Duplex Sequencing - A RAPID, ACCURATE AND MECHANISTICALLY INFORMATIVE GENOTOXICITY ASSAY USING
DUPLEX SEQUENCING
Genotoxicity testing, the evaluation of chemicals for the potential to induce DNA mutations increasing
human cancer risk, is a crucial part of development of drugs, cosmetics and other products. But the
available battery of mutagenicity assays forces frustrating trade-offs between performance and
convenience. And while existing tests can detect mutagenesis, most provide no insight into mechanism
of action. A more convenient accurate, rapid, and mechanistically-informative assay is needed to speed
drug and other chemical development and better detect threats to human health.
This SBIR grant application proposes to develop an end-to-end laboratory kit and software-based
genotoxicity testing product using Duplex Sequencing, the most accurate DNA sequencing technology
in existence. Because Duplex Sequencing can detect ultra-low frequency mutations at levels below
one-in-ten-million, it can identify a chemical as mutagenic within weeks of exposure. Because it relies
on a simple workflow that only requires extracted DNA as an input, it can be deployed far more widely
than existing in vivo assays. And because it assesses not just mutation frequency but also the identities
of the specific mutations induced, Duplex Sequencing will be the only genotoxicity test that reveals
chemicals’ trinucleotide “mutational signatures”, which can implicate specific mutagenic mechanisms,
thus enabling strategic redesign of a mutagenic but otherwise promising drug.
We have generated strong proof-of-principle results, but substantial work is needed to translate our
experimental assay into an off-the-shelf product that customers can easily adopt. In this proposal we
will optimize and validate a user-friendly Duplex Sequencing genotoxicity assay in preparation for
commercialization. In Phase I we will construct Duplex Sequencing panels for measurement of
genotoxicity in rats, mice and humans, and evaluate their performance. In Phase II we will optimize the
assay’s protocols and reagents, build and deploy cloud-based software allowing biologists to easily
render Duplex Sequencing outputs into interpretable results, and validate our assay in animal and
human cell line models over diverse mutagens, inclusive of assessing their mutational signatures.
Genotoxicity testing is a vital discipline that has relied on antiquated techniques for too long. The speed,
accuracy and information content of Duplex Sequencing promises a radical improvement to the status
quo. We expect this convenient and easy-to-implement test to find rapid uptake among drug companies
searching for better, earlier genotoxicity readouts, in CROs excited for a broadly applicable assay they
can offer their customers and among academics, looking to push the envelope of their fields.
