Abstract
To improve the accuracy and the cost-efficiency of next-generation sequencing in ultralow-frequency mutation detection, we developed the Paired-End and Complementary Consensus Sequencing (PECC-Seq), a PCR-free duplex consensus sequencing approach. PECC-Seq employed shear points as endogenous barcodes to identify consensus sequences from the overlap in the shortened, complementary DNA strand-derived paired-end reads for sequencing error correction. With the high accuracy of PECC-Seq, we identified the characteristic base substitution errors introduced by the end-repair process of mechanical fragmentation-based library preparations, which were prominent at the terminal 7 bp of the library fragments in the 5′-NpCpA-3′ and 5′-NpCpT-3′ trinucleotide context. As demonstrated at the human genome scale (TK6 cells), after removing these potential end-repair artifacts from the terminal 7 bp, PECC-Seq could reduce the sequencing error frequency to mid-10−7 with a relatively low sequencing depth. For TA base pairs, the background error rate could be suppressed to mid-10−8. In mutagen-treated (6 μg/mL methyl methanesulfonate or 12 μg/mL N-nitroso-N-ethylurea) TK6, increases in mutagen treatment-related mutant frequencies could be detected, indicating the potential of PECC-Seq in detecting genome-wide ultra-rare mutations. In addition, our finding on the patterns of end-repair artifacts may provide new insights into further reducing technical errors not only for PECC-Seq, but also for other next-generation sequencing techniques.
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Acknowledgements
We appreciate Dr. Rajaguru Palanisamy for his critical reading and suggestions on our manuscript.
Funding
This study was funded by the Project Research on Regulatory Harmonization and Evaluation of Pharmaceuticals, Medical Devices, Regenerative and Cellular Therapy Products, Gene Therapy Products, and Cosmetics from the Japan Agency for Medical Research and Development, AMED (Grant number 16mk0102010j0003).
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XY, ST and TS designed the study and performed the experiments. TS and YL supervised the study. XY analyzed and interpreted the data, and was a major contributor in writing the manuscript. MH provided the cell lines used in the study. WL, YC, MN and CF helped design the study and provided constructive suggestions during the study. All authors read and approved the final manuscript.
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The raw whole genome sequencing data used in this study are available in the NCBI Sequence Read Archive with the accession number of PRJNA632709.
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You, X., Thiruppathi, S., Liu, W. et al. Detection of genome-wide low-frequency mutations with Paired-End and Complementary Consensus Sequencing (PECC-Seq) revealed end-repair-derived artifacts as residual errors. Arch Toxicol 94, 3475–3485 (2020). https://doi.org/10.1007/s00204-020-02832-0
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DOI: https://doi.org/10.1007/s00204-020-02832-0