Science and Nature

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

Summary

Identification and quantification of low-frequency mutations remain tantalizing despite improvements in the baseline error rate of subsequent-technology sequencing applied sciences. Here, we characterize a technique, termed SaferSeqS, that addresses these challenges by (1) effectively introducing identical molecular barcodes in the Watson and Crick strands of template molecules and (2) enriching aim sequences with strand-specific PCR. The manner 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.

Recordsdata availability

The sequencing data generated on this respect will likely be obtained from the European Genome–phenome Archive (accession amount EGAS00001005048).

Code availability

The SaferSeqS bioinformatics pipeline is implemented in Python. The source code is on hand in a Zenodo repository (https://doi.org/10.5281/zenodo.4588264).

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Acknowledgements

We thank the other folks who participated on this respect for their braveness and generosity. We also thank M. Hoang, S. Sur, A. Mattox, A. Pearlman and members of the Ludwig Heart at Johns Hopkins for insightful and priceless scientific discussions. We’re grateful to C. Blair and Okay. Deem for educated technical and administrative assistance and to E. Cook dinner for illustrative assistance. This work was supported by The Lustgarten Foundation for Pancreatic Cancer Research, The Marcus Foundation, The Virginia and D.Okay. 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).

Creator data

Affiliations

  1. Ludwig Heart for Cancer Genetics and Therapeutics, Johns Hopkins College Faculty of Medication, Baltimore, MD, USA

    Joshua D. Cohen, Christopher Douville, Jonathan C. Dudley, Brian J. Mog, Maria Popoli, Janine Ptak, Lisa Dobbyn, Natalie Silliman, Joy Schaefer, Nickolas Papadopoulos, Kenneth W. Kinzler & Bert Vogelstein

  2. Sidney Kimmel Complete Cancer Heart, Johns Hopkins College Faculty of Medication, Baltimore, MD, USA

    Joshua D. Cohen, Christopher Douville, Jonathan C. Dudley, Brian J. Mog, Maria Popoli, Janine Ptak, Lisa Dobbyn, Natalie Silliman, Joy Schaefer, Cristian Tomasetti, Nickolas Papadopoulos, Kenneth W. Kinzler & Bert Vogelstein

  3. Sol Goldman Pancreatic Cancer Research Heart, Johns Hopkins College Faculty of Medication, Baltimore, MD, USA

    Joshua D. Cohen, Christopher Douville, Jonathan C. Dudley, Brian J. Mog, Maria Popoli, Janine Ptak, Lisa Dobbyn, Natalie Silliman, Joy Schaefer, Nickolas Papadopoulos, Kenneth W. Kinzler & Bert Vogelstein

  4. Howard Hughes Medical Institute, Baltimore, MD, USA

    Joshua D. Cohen, Christopher Douville, Jonathan C. Dudley, Brian J. Mog, Maria Popoli, Janine Ptak, Natalie Silliman & Bert Vogelstein

  5. Division of Biomedical Engineering, Johns Hopkins College, Baltimore, MD, USA

    Joshua D. Cohen & Brian J. Mog

  6. Division of Personalized Oncology, Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia

    Jeanne Tie & Peter Gibbs

  7. Division of Medical Oncology, Peter MacCallum Cancer Heart, Melbourne, Victoria, Australia

    Jeanne Tie

  8. Division of Medical Oncology, Western Health, Melbourne, Victoria, Australia

    Jeanne Tie & Peter Gibbs

  9. Faculty of Medication, Dentistry and Health Sciences, College of Melbourne, Melbourne, Victoria, Australia

    Jeanne Tie & Peter Gibbs

  10. Division of Biostatistics, Johns Hopkins Bloomberg Faculty of Public Health, Baltimore, MD, USA

    Cristian Tomasetti

Contributions

J.D.C., N.P., Okay.W.Okay. and B.V. conceptualized the SaferSeqS method. J.D.C., C.D., J.C.D., B.J.M., N.P., Okay.W.Okay. and B.V. contributed to the respect fabricate. J.D.C., M.P., J.P., L.D., N.S. and J.S. performed the experiments. J.T. and P.G. recruited contributors and obtained samples. J.D.C. developed the SaferSeqS bioinformatic pipeline and analyzed the data. Mathematical and statistical analyses had been performed by J.D.C. and C.T. N.P., Okay.W.Okay. and B.V. supervised the respect. J.D.C. and B.V. wrote the manuscript, which was edited and popular by all authors.

Corresponding authors

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

Ethics declarations

Competing pursuits

B.V., Okay.W.Okay. and N.P. are founders of Thrive and Private Genome Diagnostics and maintain equity in Staunch Sciences and Private Genome Diagnostics. Okay.W.Okay. and N.P. are consultants to Thrive. Okay.W.Okay. and B.V. are consultants to Sysmex and Eisai, and Okay.W.Okay., B.V. and N.P. are advisors to CAGE Pharma. B.V. is also a well informed to Catalio, and Okay.W.Okay., B.V. and N.P. are consultants to Neophore. C.D. is a well informed to Thrive and is compensated with profits and equity. The companies named above, as well to barely a kind of companies, have licensed previously described applied sciences linked to the work described on this paper from Johns Hopkins College. J.D.C., C.D., B.V., Okay.W.Okay., C.T. and N.P. are inventors on all these applied sciences. Licenses to these applied sciences are or will likely be linked to equity or royalty payments to the inventors as well to to Johns Hopkins College. Additional patent capabilities on the work described on this paper are being filed by Johns Hopkins College. The terms of all these arrangements are being managed by Johns Hopkins College in step with its war of interest insurance policies. The final authors uncover no competing pursuits.

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Witness overview data Nature Biotechnology thanks Paul Spellman and the barely a kind of, anonymous, reviewer(s) for their contribution to the label overview of this work.

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Cohen, J.D., Douville, C., Dudley, J.C. et al. Detection of low-frequency DNA variants by centered sequencing of the Watson and Crick strands.
Nat Biotechnol (2021). https://doi.org/10.1038/s41587-021-00900-z

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