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Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands


Identification and quantification of low-frequency mutations remain challenging despite improvements in the baseline error rate of next-generation sequencing technologies. Here, we describe a method, termed SaferSeqS, that addresses these challenges by (1) efficiently introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching target sequences with strand-specific PCR. The method achieves high sensitivity and specificity and detects variants at frequencies below 1 in 100,000 DNA template molecules with a background mutation rate of <5 × 10–7 mutants per base pair (bp). We demonstrate that it can evaluate mutations in a single amplicon or simultaneously in multiple amplicons, assess limited quantities of cell-free DNA with high recovery of both strands and reduce the error rate of existing PCR-based molecular barcoding approaches by >100-fold.

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Fig. 1: Overview of SaferSeqS.
Fig. 2: Detection of mutations in liquid biopsy samples.
Fig. 3: Multiplex panel for the detection of cancer driver gene mutations.
Fig. 4: Clinical application of the SaferSeqS multiplex panel.

Data availability

The sequencing data generated in this study can be obtained from the European Genome–phenome Archive (accession number EGAS00001005048).

Code availability

The SaferSeqS bioinformatics pipeline is implemented in Python. The source code is available in a Zenodo repository (


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We thank the individuals who participated in this study for their courage and generosity. We also thank M. Hoang, S. Sur, A. Mattox, A. Pearlman and members of the Ludwig Center at Johns Hopkins for insightful and helpful scientific discussions. We are grateful to C. Blair and K. Judge for expert technical and administrative assistance and to E. Cook for illustrative assistance. This work was supported by The Lustgarten Foundation for Pancreatic Cancer Research, The Marcus Foundation, The Virginia and D.K. Ludwig Fund for Cancer Research, The Conrad N. Hilton Foundation, The John Templeton Foundation, Medical Research Future Fund Investigator Grant (APP1194970) and National Institutes of Health grants (T32 GM007309, U01 CA230691-01, P50 CA228991, U01 CA200469, R37 CA230400-01, and U01 CA152753).

Author information




J.D.C., N.P., K.W.K. and B.V. conceptualized the SaferSeqS method. J.D.C., C.D., J.C.D., B.J.M., N.P., K.W.K. and B.V. contributed to the study design. J.D.C., M.P., J.P., L.D., N.S. and J.S. performed the experiments. J.T. and P.G. recruited participants and acquired samples. J.D.C. developed the SaferSeqS bioinformatic pipeline and analyzed the data. Mathematical and statistical analyses were conducted by J.D.C. and C.T. N.P., K.W.K. and B.V. supervised the study. J.D.C. and B.V. wrote the manuscript, which was edited and approved by all authors.

Corresponding authors

Correspondence to Nickolas Papadopoulos or Kenneth W. Kinzler or Bert Vogelstein.

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Competing interests

B.V., K.W.K. and N.P. are founders of Thrive and Personal Genome Diagnostics and own equity in Exact Sciences and Personal Genome Diagnostics. K.W.K. and N.P. are consultants to Thrive. K.W.K. and B.V. are consultants to Sysmex and Eisai, and K.W.K., B.V. and N.P. are advisors to CAGE Pharma. B.V. is also a consultant to Catalio, and K.W.K., B.V. and N.P. are consultants to Neophore. C.D. is a consultant to Thrive and is compensated with income and equity. The companies named above, as well as other companies, have licensed previously described technologies related to the work described in this paper from Johns Hopkins University. J.D.C., C.D., B.V., K.W.K., C.T. and N.P. are inventors on some of these technologies. Licenses to these technologies are or will be associated with equity or royalty payments to the inventors as well as to Johns Hopkins University. Additional patent applications on the work described in this paper are being filed by Johns Hopkins University. The terms of all these arrangements are being managed by Johns Hopkins University in accordance with its conflict of interest policies. The remaining authors declare no competing interests.

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Peer review information Nature Biotechnology thanks Paul Spellman and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Cohen, J.D., Douville, C., Dudley, J.C. et al. Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands. Nat Biotechnol (2021).

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