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

Nature Biomedical Engineering volume 6, pages 257–266 (2022)Cite this article

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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.

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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.

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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.

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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.).

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Author notes

  1. These authors contributed equally: Gregory Gydush, Erica Nguyen.

Authors and Affiliations

  1. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Gregory Gydush, Erica Nguyen, Jin H. Bae, Timothy Blewett, Justin Rhoades, Douglas Shea, Kan Xiong, Ruolin Liu, Atish D. Choudhury, J. Christopher Love, Todd R. Golub & Viktor A. Adalsteinsson

  2. Vanderbilt University, Nashville, TN, USA

    Sarah C. Reed

  3. Department of Radiation Oncology, Dana-Farber Cancer Institute and Brigham and Women’s Hospital, Boston, MA, USA

    Fangyan Yu, Ka Wai Leong & G. Mike Makrigiorgos

  4. Harvard Medical School, Boston, MA, USA

    Ka Wai Leong, Atish D. Choudhury & Todd R. Golub

  5. Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA, USA

    Atish D. Choudhury, Sara M. Tolaney, Ian E. Krop, Heather A. Parsons & Todd R. Golub

  6. Ohio State University Comprehensive Cancer Center, Columbus, OH, USA

    Daniel G. Stover

  7. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, USA

    J. Christopher Love & Viktor A. Adalsteinsson

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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.

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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.

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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

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