The October 2021 EMM Editor’s Choice article is “PacBio sequencing detects genome-wide ultra-low-frequency substitution mutations resulting from exposure to chemical mutagens”(https://onlinelibrary.wiley.com/doi/full/10.1002/em.22462) by Javier R. Revollo, Jaime A. Miranda, and Vasily N. Dobrovolsky.
The progress in next-generation sequencing (NGS) technology is leading revolutionary changes in numerous research fields, e.g., clinical medicine, drug development, toxicology, and risk assessment. However, an error rate between 0.1% and 1% among NGS techniques has hindered its application in low-frequency mutation detection, especially for the whole genome sequencing. Researchers (Javier R. Revollo, Jaime A. Miranda, and Vasily N. Dobrovolsky) at the National Center for Toxicological Research have optimized the PacBio Single-Molecule, Real-Time (PB SMRT) sequencing and successfully measure rare substitution mutations in E. coli cultures exposed to ethyl methanosulfonate (EMS) or N-ethyl-N-nitrosourea (ENU). PacBio sequencing captures the long nucleotide sequence from a single DNA molecule where a DNA polymerase performing uninterrupted template-directed synthesis using four fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). This new technology provides a better opportunity to identify rare mutations across entire genomes. In this study, Revollo et al. carried out a study to improve the accuracy of PacBio sequencing for the genome-wide mutation detection by optimizing the consensus mode (“single” versus “forward and reverse”), RQ filtering criteria and library size. The optimized method achieves high sensitivity and detects ultra-low-frequency substitution mutations in E. coli cultures with a background mutation rate of <1 × 10–7 mutants per base pair. In addition, Revollo et al. demonstrated the ability of PB SMRT sequencing for detecting a concentration-dependent increase in the per-nucleotide mutant frequency, induced by a mutagen, ENU. Mutant frequencies and spectra observed by PB SMRT are comparable to those obtained by clone-sequencing from the same exposures. Taken together, this study proved the power of PB SMRT sequencing in genome-wide quantification of rare substitution mutations. The described PB SMRT sequencing method could be useful for mutational research and regulatory safety testing by providing an alternative approach to Illumina-based error-corrected NGS techniques.