Abstract
The efficiency of DNA-enrichment techniques is often insufficient to detect mutations that occur at low frequencies. Here we report a DNA-excision method for the detection of low-frequency mutations in genomic DNA and in circulating cell-free DNA at single-nucleotide resolution. The method is based on a competitive DNA-binding-and-digestion mechanism, effected by deoxyribonuclease I (DNase) guided by single-stranded phosphorothioated DNA (sgDNase), for the removal of wild-type DNA strands. The sgDNase can be designed against any wild-type DNA sequences, allowing for the uniform enrichment of all the mutations within the target-binding region of single-stranded phosphorothioated DNA at mild-temperature conditions. Pretreatment with sgDNase enriches all mutant strands with initial frequencies down to 0.01% and leads to high discrimination factors for all types of single-nucleotide mismatch in multiple sequence contexts, as we show for the identification of low-abundance mutations in samples of blood or tissue from patients with cancer. The method can be coupled with next-generation sequencing, droplet digital polymerase chain reaction, Sanger sequencing, fluorescent-probe-based assays and other mutation-detection methods.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. All data generated during the study are available from figshare with the identifier https://doi.org/10.6084/m9.figshare.23529204. Source data for the figures are provided with this paper.
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Acknowledgements
We thank the National Center for Protein Science at Peking University (Beijing, China) for assistance with droplet digital PCR. This work was supported by the National Natural Science Foundation of China (22174005, 21974005, 21575008).
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M. Zhao conceived the project. M. Zhao, W.C., T.W. and X.X. performed the sequences and experiments design. W.C., H.X. and S.D. conducted the experiments and data analysis. J.W., Z.Y., Y.J. and M. Zou helped improve some experiments. Z.L., W.Y. and B.Z. provided blood and tissue samples from patients with venous malformations. X.X. provided blood and tissue samples from patients with NSCLC or colorectal cancer. W.C., T.W. and M. Zhao wrote the manuscript.
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Peking University holds an issued Chinese patent (patent number: ZL 2019 1 0052680.4) on this work. The authors declare no other competing interests.
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Nature Biomedical Engineering thanks Feng Li, David Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available.
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Extended data
Extended Data Fig. 1 Multiplexed sgDNase pretreatment.
Next-generation sequencing results of the five EGFR and KRAS mutations (EGFR L858R, EGFR 19del, EGFR G719S, EGFR T790M and KRAS G13D) in mixed synthetic samples before (a1-a5) and after (b1-b5) 5-plex sgDNase pretreatment. The initial mutation abundance was 0.4% for EGFR G719S and 0.1% for other tested mutation. The five target mutations were pretreated by sgDNase simultaneously. The variant to noise plots derived from Illumina MiSeq sequencing data are depicted.
Extended Data Fig. 2 Detection of low-abundance mutations in clinical samples with sgDNase pretreatment.
NGS measurement results of the abundance of EGFR L858R mutation in a gDNA (P2-T) and b cfDNA (P2-B) without sgDNase pretreatment. Variant to noise plots derived from Illumina MiSeq sequencing data are depicted.
Extended Data Fig. 3 Summary of sample test results with sgDNase pretreatment.
a, Beeswarm plot of post-VAFs of 309 negative control gDNA samples and 267 positive samples with pre-VAF at 0.01% after sgDNase pretreatment. b, Receiver operator characteristic (ROC) plot for post-VAFs of samples in a after pretreatment by sgDNase.
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Chen, W., Xu, H., Dai, S. et al. Detection of low-frequency mutations in clinical samples by increasing mutation abundance via the excision of wild-type sequences. Nat. Biomed. Eng 7, 1602–1613 (2023). https://doi.org/10.1038/s41551-023-01072-8
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DOI: https://doi.org/10.1038/s41551-023-01072-8
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