Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Massively parallel enrichment of low-frequency alleles enables duplex sequencing at low depth

Abstract

Assaying for large numbers of low-frequency mutations requires sequencing at extremely high depth and accuracy. Increasing sequencing depth aids the detection of low-frequency mutations yet limits the number of loci that can be simultaneously probed. Here we report a method for the accurate tracking of thousands of distinct mutations that requires substantially fewer reads per locus than conventional hybrid-capture duplex sequencing. The method, which we named MAESTRO (for minor-allele-enriched sequencing through recognition oligonucleotides), combines massively parallel mutation enrichment with duplex sequencing to track up to 10,000 low-frequency mutations, with up to 100-fold fewer reads per locus. We show that MAESTRO can be used to test for chimaerism by tracking donor-exclusive single-nucleotide polymorphisms in sheared genomic DNA from human cell lines, to validate whole-exome sequencing and whole-genome sequencing for the detection of mutations in breast-tumour samples from 16 patients, and to monitor the patients for minimal residual disease via the analysis of cell-free DNA from liquid biopsies. MAESTRO improves the breadth, depth, accuracy and efficiency of mutation testing by sequencing.

This is a preview of subscription content, access via your institution

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Fig. 1: MAESTRO enables accurate mutation tracking using minimal sequencing in clinical specimens.
Fig. 2: MAESTRO uncovers most mutant duplexes using substantially fewer reads.
Fig. 3: MAESTRO fingerprint validation of whole exome tumour samples.
Fig. 4: MAESTRO can detect signal above noise at 1:100,000 dilution.
Fig. 5: MAESTRO improves detection of MRD in pre-operative setting.

Data availability

The data supporting the results in this study are available within the paper and its Supplementary Information. Sequencing data have been deposited into the controlled-access database Data Use Oversight System (DUOS; http://duos.broadinstitute.org) under the accession number DUOS-000135.

Code availability

Custom code for designing MAESTRO probes from somatic variant calls can be found at https://github.com/broadinstitute/MAESTRO-probe_designer.

References

  1. Luquette, L. J., Bohrson, C. L., Sherman, M. A. & Park, P. J. Identification of somatic mutations in single cell DNA-seq using a spatial model of allelic imbalance. Nat. Commun. 10, 3908 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  2. Ludwig, L. S. et al. Lineage tracing in humans enabled by mitochondrial mutations and single-cell genomics. Cell 176, e22 (2019).

    Article  Google Scholar 

  3. Zahn, L. M. Mapping genotype to phenotype. Science 362, 555.4–556 (2018).

    Article  Google Scholar 

  4. D’Gama, A. M. & Walsh, C. A. Somatic mosaicism and neurodevelopmental disease. Nat. Neurosci. 21, 1504–1514 (2018).

    Article  PubMed  Google Scholar 

  5. Garcia-Murillas, I. et al. Assessment of molecular relapse detection in early-stage breast cancer. JAMA Oncol. https://doi.org/10.1001/jamaoncol.2019.1838 (2019).

  6. Canick, J. A., Palomaki, G. E., Kloza, E. M., Lambert-Messerlian, G. M. & Haddow, J. E. The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat. Diagn. 33, 667–674 (2013).

    Article  CAS  PubMed  Google Scholar 

  7. Bejar, R. et al. Somatic mutations predict poor outcome in patients with myelodysplastic syndrome after hematopoietic stem-cell transplantation. J. Clin. Oncol. 32, 2691–2698 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Snyder, T. M., Khush, K. K., Valantine, H. A. & Quake, S. R. Universal noninvasive detection of solid organ transplant rejection. Proc. Natl Acad. Sci. USA 108, 6229–6234 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Blauwkamp, T. A. et al. Analytical and clinical validation of a microbial cell-free DNA sequencing test for infectious disease. Nat. Microbiol 4, 663–674 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Boyd, S. D. et al. Measurement and clinical monitoring of human lymphocyte clonality by massively parallel VDJ pyrosequencing. Sci. Transl. Med. 1, 12ra23 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Gilbert, J. A. et al. Current understanding of the human microbiome. Nat. Med. 24, 392–400 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lowe, A., Murray, C., Whitaker, J., Tully, G. & Gill, P. The propensity of individuals to deposit DNA and secondary transfer of low level DNA from individuals to inert surfaces. Forensic Sci. Int. 129, 25–34 (2002).

    Article  CAS  PubMed  Google Scholar 

  13. Schmitt, M. W. et al. Detection of ultra-rare mutations by next-generation sequencing. Proc. Natl Acad. Sci. USA 109, 14508–14513 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Song, C. et al. Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment. Nucleic Acids Res. 44, e146 (2016).

    PubMed  PubMed Central  Google Scholar 

  15. Li, J. & Makrigiorgos, G. Mike COLD-PCR: a new platform for highly improved mutation detection in cancer and genetic testing. Biochem. Soc. Trans. 37, 427–432 (2009).

    Article  CAS  PubMed  Google Scholar 

  16. Wu, L. R., Chen, S. X., Wu, Y., Patel, A. A. & Zhang, D. Y. Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification. Nat. Biomed. Eng. 1, 714–723 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Jeffreys, A. J. & May, C. A. DNA enrichment by allele-specific hybridization (DEASH): a novel method for haplotyping and for detecting low-frequency base substitutional variants and recombinant DNA molecules. Genome Res. 13, 2316–2324 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Gaudet, M., Fara, A.-G., Beritognolo, I. & Sabatti, M. Allele-specific PCR in SNP genotyping. Methods Mol. Biol. 578, 415–424 (2009).

    Article  CAS  PubMed  Google Scholar 

  19. Vargas, D. Y., Marras, S. A. E., Tyagi, S. & Kramer, F. R. Suppression of wild-type amplification by selectivity enhancing agents in PCR assays that utilize superselective primers for the detection of rare somatic mutations. J. Mol. Diagn. 20, 415–427 (2018).

    Article  CAS  PubMed  Google Scholar 

  20. Li, J. et al. Replacing PCR with COLD-PCR enriches variant DNA sequences and redefines the sensitivity of genetic testing. Nat. Med. 14, 579–584 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Li, J., Milbury, C. A., Li, C. & Makrigiorgos, G. M. Two-round coamplification at lower denaturation temperature-PCR (COLD-PCR)-based sanger sequencing identifies a novel spectrum of low-level mutations in lung adenocarcinoma. Hum. Mutat. 30, 1583–1590 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Schmitt, M. W. et al. Sequencing small genomic targets with high efficiency and extreme accuracy. Nat. Methods 12, 423–425 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Parsons, H. A. et al. Sensitive detection of minimal residual disease in patients treated for early-stage breast cancer. Clin. Cancer Res. https://doi.org/10.1158/1078-0432.CCR-19-3005 (2020).

  24. Newman, A. M. et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat. Biotechnol. 34, 547–555 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Machin, G. Non-identical monozygotic twins, intermediate twin types, zygosity testing, and the non-random nature of monozygotic twinning: a review. Am. J. Med. Genet. C 151C, 110–127 (2009).

    Article  Google Scholar 

  26. Shimoni, A. & Nagler, A. Non-myeloablative stem cell transplantation (NST): chimerism testing as guidance for immune-therapeutic manipulations. Leukemia 15, 1967–1975 (2001).

    Article  CAS  PubMed  Google Scholar 

  27. Breuer, S. et al. Early recipient chimerism testing in the T- and NK-cell lineages for risk assessment of graft rejection in pediatric patients undergoing allogeneic stem cell transplantation. Leukemia 26, 509–519 (2012).

    Article  CAS  PubMed  Google Scholar 

  28. Tyler, J., Kumer, L., Fisher, C., Casey, H. & Shike, H. Personalized chimerism test that uses selection of short tandem repeat or quantitative PCR depending on patient’s chimerism status. J. Mol. Diagn. 21, 483–490 (2019).

    Article  CAS  PubMed  Google Scholar 

  29. Newman, A. M. et al. An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat. Med. 20, 548–554 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Lee, H., Park, C., Na, W., Park, K. H. & Shin, S. Precision cell-free DNA extraction for liquid biopsy by integrated microfluidics. npj Precis. Oncol. 4, 3 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Mauger, F. et al. Comparison of commercially available whole-genome sequencing kits for variant detection in circulating cell-free DNA. Sci. Rep. 10, 6190 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Liu, D. et al. Multiplex cell-free DNA reference materials for quality control of next-generation sequencing-based in vitro diagnostic tests of colorectal cancer tolerance. J. Cancer 9, 3812–3823 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tsao, D. S. et al. A novel high-throughput molecular counting method with single base-pair resolution enables accurate single-gene NIPT. Sci. Rep. 9, 14382 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  34. Pantel, K. & Alix-Panabières, C. Liquid biopsy and minimal residual disease—latest advances and implications for cure. Nat. Rev. Clin. Oncol. 16, 409–424 (2019).

    Article  CAS  PubMed  Google Scholar 

  35. Tie, J. et al. Circulating tumor DNA analysis detects minimal residual disease and predicts recurrence in patients with stage II colon cancer. Sci. Transl. Med. 8, 346ra92 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  36. Chaudhuri, A. A. et al. Early detection of molecular residual disease in localized lung cancer by circulating tumor DNA profiling. Cancer Discov. 7, 1394–1403 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Coombes, R. C. et al. Personalized detection of circulating tumor DNA antedates breast cancer metastatic recurrence. Clin. Cancer Res. 25, 4255–4263 (2019).

    Article  CAS  PubMed  Google Scholar 

  38. Wan, J. C. M. et al. ctDNA monitoring using patient-specific sequencing and integration of variant reads. Sci. Transl. Med. 12, eaaz8084 (2020).

    Article  CAS  PubMed  Google Scholar 

  39. McDonald, B. R. et al. Personalized circulating tumor DNA analysis to detect residual disease after neoadjuvant therapy in breast cancer. Sci. Transl. Med. 11, eaax7392 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  40. Butler, T. M. et al. Circulating tumor DNA dynamics using patient-customized assays are associated with outcome in neoadjuvantly treated breast cancer. Cold Spring Harb. Mol. Case Stud. 5, a003772 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Magbanua, M. J. M. et al. Circulating tumor DNA in neoadjuvant treated breast cancer reflects response and survival. Oncology https://doi.org/10.1101/2020.02.03.20019760 (2020).

  42. Moding, E. J. et al. Circulating tumor DNA dynamics predict benefit from consolidation immunotherapy in locally advanced non-small-cell lung cancer. Nat. Cancer 1, 176–183 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Etienne, G. et al. Long-term follow-up of the French Stop Imatinib (STIM1) study in patients with chronic myeloid leukemia. J. Clin. Oncol. 35, 298–305 (2017).

    Article  PubMed  Google Scholar 

  44. Abbosh, C. et al. Phylogenetic ctDNA analysis depicts early-stage lung cancer evolution. Nature 545, 446–451 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Zviran, A. et al. Genome-wide cell-free DNA mutational integration enables ultra-sensitive cancer monitoring. Nat. Med. 26, 1114–1124 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Masuda, N. et al. Adjuvant capecitabine for breast cancer after preoperative chemotherapy. N. Engl. J. Med. 376, 2147–2159 (2017).

    Article  CAS  PubMed  Google Scholar 

  47. von Minckwitz, G. et al. Trastuzumab emtansine for residual invasive HER2-positive breast cancer. N. Engl. J. Med. 380, 617–628 (2019).

    Article  Google Scholar 

  48. Zook, J. M. et al. An open resource for accurately benchmarking small variant and reference calls. Nat. Biotechnol. 37, 561–566 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Genomes Project Consortium et al. A global reference for human genetic variation. Nature 526, 68–74 (2015). 1000.

    Article  Google Scholar 

  50. Adalsteinsson, V. A. et al. Scalable whole-exome sequencing of cell-free DNA reveals high concordance with metastatic tumors. Nat. Commun. 8, 1324 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  51. Köster, J. & Rahmann, S. Snakemake—a scalable bioinformatics workflow engine. Bioinformatics 28, 2520–2522 (2012).

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We acknowledge the patients and their families for their contributions to this study. We also thank the generous support from the Gerstner Family Foundation. This project was supported in part by the Bridge Project (J.C.L. and V.A.A.), a partnership between the Koch Institute for Integrative Cancer Research at MIT and the Dana-Farber/Harvard Cancer Center. We also acknowledge support from National Institutes of Health grants R33 CA217652 (G.M.M. and V.A.A.) and R01 CA221874 (G.M.M. and V.A.A.).

Author information

Authors and Affiliations

Authors

Contributions

G.G., E.N. and J.H.B. designed the research, analysed the data and wrote the manuscript. T.B. and J.R. analysed the data and created figures for the manuscript. S.C.R., D.S., K.X., R.L., F.Y. and K.W.L. designed experiments and interpreted the results. A.D.C., D.G.S., S.M.T., I.E.K. and H.A.P. contributed clinical samples and the interpretation of results. J.C.L., G.M.M., T.R.G. and V.A.A. designed the research, interpreted the results and wrote the manuscript. All authors reviewed and approved the final version of the manuscript.

Corresponding authors

Correspondence to G. Mike Makrigiorgos, Todd R. Golub or Viktor A. Adalsteinsson.

Ethics declarations

Competing interests

A.D.C. has advisory-board roles with Clovis, Dendreon and Bayer, and has received research funding from Bayer. S.M.T. has received research funding to the institution from AstraZeneca, Eli Lilly, Merck, Novartis, Nektar, Pfizer, Genentech, Immunomedics, Exelixis, Bristol-Myers Squibb, Eisai, Nanostring, Cyclacel, Sanofi, Odonate and Seattle Genetics, and has received honorariums for consulting and advisory-board participation from AstraZeneca, Eli Lilly, Merck, Novartis, Nektar, Pfizer, Genentech, Immunomedics, Bristol-Myers Squibb, Eisai, Nanostring, Sanofi, Odonate, Seattle Genetics, Puma, Anthenex, OncoPep, Abbvie, G1 Therapeutics, Silverback Therapeutics and Celldex. I.E.K. has received research funding to the institute from Genentech, Pfizer and Daichii-Sankyo, and has received honorariums for consulting and advisory-board participation from Genentech, Daichii-Sankyo, Macrogenics, Context Therapeutics, Taiho Oncology, Merck, Novartis and Bristol-Myers Squibb. H.A.P. has a paid consultant role for Foundation Medicine. T.R.G. is a paid advisor to GlaxoSmithKline, and is a co-founder and equity holder of Sherlock Biosciences and FORMA Therapeutics. V.A.A. has a patent application filed with the Broad Institute, and is a member of the scientific advisory boards of Bertis and AGCT, which were not involved in this study. The other authors report no competing interests.

Peer review

Peer review information

Nature Biomedical Engineering thanks the anonymous reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary figures.

Reporting Summary

Supplementary Data 1

Probe sequences.

Supplementary Data 2

Supplementary tables.

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Gydush, G., Nguyen, E., Bae, J.H. et al. Massively parallel enrichment of low-frequency alleles enables duplex sequencing at low depth. Nat. Biomed. Eng 6, 257–266 (2022). https://doi.org/10.1038/s41551-022-00855-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41551-022-00855-9

Search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer