Abstract
Error-corrected next generation sequencing (ecNGS) is attracting attention as a new technique for genotoxicity testing. It is a method that can detect low-frequency mutations through direct sequencing without relying on phenotypes, by reducing the error of the NGS to the ultimate level. Traditionally, in vivo mutation tests have been performed using transgenic rodents (TGR) in which target genes have been introduced. ecNGS is applicable to all species, including humans, and is expected to replace these tests in the near future. In fact, the OECD TGR test guideline (TG488) is currently undergoing accelerated movement toward revision.
In Europe and the U.S., the Duplex Sequencing method has already become popular as ecNGS, and the Nano-Seq and HiFi-Seq methods have also been developed. In Japan, our original PECC-Seq, and the HAWK-seq are also developed. The characteristics and current status of these ecNGS methods will be introduced.
The ecNGS method can be applied to all in vitro assays, including bacteria, and allows comparison of in vitro and in vivo test results using a common indicator. Conventional in vitro genotoxicity tests have high sensitivity but difficulty in quantitative evaluation. With ecNGS, direct comparison with in vivo test results can lead to the development of more quantitative in vitro tests. The development of test systems using novel human cells such as organoids and MPS combined with the ecNGS method is already underway, and it is hoped that this will lead to the development of test systems that enable the evaluation of real effects in humans. The ecNGS method can be directly incorporated into general toxicity studies such as 28-day repeated-dose tests in rodents, and is expected to be widely used as a multi-endpoint toxicity test that incorporates genotoxicity testing into repeated-dose studies in the near future.
