Application of duplex sequencing to evaluate mutagenicity -Pub

I became interested in the topic of alternative in vivo assays after hearing @conudel. Later, I saw a fantastic presentation on this topic at the FDA/HESI Workshop last year.

Today, I bring you a publication by Tao et al. from the Division of Genetic and Molecular Toxicology in the FDA on ‘Application of duplex sequencing to evaluate mutagenicity of aristolochic acid and methapyrilene in Fisher 344 rats’

Duplex sequencing (DS) is an error-corrected next-generation sequencing (NGS) method that can overcome notorious high error rate from the process of NGS and detect ultralow-frequency mutations. In this study, we evaluated the mutagenicity of aristolochic acid, a known genotoxic carcinogen, and methapyrilene, a known nongenotoxic carcinogen using DS. Four male Fisher 344 rats were treated with aristolochic acid, methapyrilene, or the vehicle control for 6 weeks, liver tissues were collected one day after the treatment, and the DNA was isolated for analysis. The mutation frequency for the aristolochic acid-treated group was significantly increased over the vehicle control (44-fold), whereas no significant difference in the mutation frequency was observed between the methapyrilene-treated and the control groups. The primary type of mutation induced by aristolochic acid was A:T > T:A transversion, which occurred frequently at ApT sites, whereas the major type of mutation in the control and methapyrilene-treated groups was G:C > A:T transition, which occurred frequently at CpG sites.
These findings are consistent with previously published data obtained with other in vivo mutation assays. Thus, our results suggest that the DS mutation assay is a promising technology for assessing mutagenicity of chemicals in vivo.


Consider yourself warned!!

Smith-Roe et al. Adopting duplex sequencing technology for genetic toxicity testing: A proof-of-concept mutagenesis experiment with N-ethyl-N-nitrosourea (ENU)-exposed rats

  • Duplex Sequencing is an ecNGS technology that directly quantifies mutations.
  • ENU-dependent mutagenesis was clearly evident at 7 d in all tissues that were tested.
  • Multiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposure.
  • Results obtained with Duplex Sequencing were highly concordant between laboratories.
  • The DuplexSeq™ Mutagenesis Assay is promising for genetic toxicity testing.

Adopting duplex sequencing technology for genetic toxicity testing_ A proof-of-concept mutagenesis experiment with N-ethyl-N-nitrosourea (ENU)-exposed rats.pdf (3.6 MB)

Schmitt et al. Detection of ultra-rare mutations bynext-generation sequencing

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 in the same position. In contrast, PCRor sequencing errors result in mutations in only one strand and can thus be discounted as a technical error. 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 cells

schmitt-et-al-2012-detection-of-ultra-rare-mutations-by-next-generation-sequencing.pdf (881.8 KB)

Polymerase Chain Reaction (PCR) Fact Sheet