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Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification

Nature Biomedical Engineering volume 1, pages 714–723 (2017)Cite this article

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A Publisher Correction to this article was published on 22 November 2017

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Abstract

Rare DNA-sequence variants hold important clinical and biological information, but existing detection techniques are expensive, complex, allele-specific, or don’t allow for significant multiplexing. Here, we report a temperature-robust polymerase-chain-reaction method, which we term blocker displacement amplification (BDA), that selectively amplifies all sequence variants, including single-nucleotide variants (SNVs), within a roughly 20-nucleotide window by 1,000-fold over wild-type sequences. This allows for easy detection and quantitation of hundreds of potential variants originally at ≤0.1% in allele frequency. BDA is compatible with inexpensive thermocycler instrumentation and employs a rationally designed competitive hybridization reaction to achieve comparable enrichment performance across annealing temperatures ranging from 56 °C to 64 °C. To show the sequence generality of BDA, we demonstrate enrichment of 156 SNVs and the reliable detection of single-digit copies. We also show that the BDA detection of rare driver mutations in cell-free DNA samples extracted from the blood plasma of lung-cancer patients is highly consistent with deep sequencing using molecular lineage tags, with a receiver operator characteristic accuracy of 95%.

The economical and high-throughput detection and quantitation of nucleic acid sequence variants is a key goal on the road to widespread adoption of precision medicine, wherein optimal individualized treatment is provided to each patient based on their unique genetic and disease profile. Profiling rare nucleic acid variants with low allele frequencies, such as cancer mutations in cell-free DNA1,2,3,4 or drug resistance in pathogen sub-populations5,6,7, presents a challenge for current molecular diagnostic technologies8. Allele-specific PCR methods9,10,11,12,13 are difficult to scale to allow highly multiplexed rare variant detection, and deep sequencing approaches14,15,16 are not economical because they waste a large majority of their read capacity on sequencing wild-type (WT) templates and amplicons. Sample enrichment to elevate the allele fractions of rare variants can allow economical sequencing-based rare variant profiling, but has been difficult to realize in highly multiplexed settings.

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Fig. 1: Temperature-robust enrichment of rare alleles by blocker displacement amplification (BDA).
Fig. 2: Quantitative PCR results on BDA enrichment.
Fig. 3: Temperature robustness of BDA.
Fig. 4: Multiplex BDA assays.
Fig. 5: Hotspot multiplexing.
Fig. 6: Rare allele quantitation with BDA.

Change history

  • 22 November 2017

    In the version of this Article originally published, owing to a technical error, the Life Sciences Reporting Summary was not included; this summary is now available.

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Acknowledgements

The authors thank J. H. Bae and J. S. Wang for experimental advice; C. Lee and G. Bao for advice and for use of their ddPCR instrument; D. Khodakov for initial testing of BDA on the MiniPCR platform; P. Song for assistance with gel electrophoresis; and A. Narayan for testing BDA in A.A.P.'s lab at the Yale School of Medicine. This work was funded by CPRIT grant RP140132 and NIH grant R01CA203964 to D.Y.Z., and NIH grant R01CA197486 to A.A.P.

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Authors and Affiliations

  1. Department of Bioengineering, Rice University, Houston, TX, 77030, USA

    Lucia R. Wu, Sherry X. Chen & David Yu Zhang

  2. Thermo Fisher, San Francisco, CA, 94080, USA

    Yalei Wu

  3. Department of Therapeutic Radiology, Yale School of Medicine, New Haven, CT, 06510, USA

    Abhijit A. Patel

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Contributions

L.R.W. conceived the project, designed and conducted the experiments, analysed the data, and wrote the paper. S.X.C. designed the experiments, wrote automated design software, and analysed the data. Y.W. conducted the experiments and analysed the data. A.A.P. provided clinical samples and analysed the data. D.Y.Z. conceived the project, guided experimental design, analysed the data, and wrote the paper.

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Correspondence to David Yu Zhang.

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Competing interests

There is a patent pending on the BDA system described in this work. D.Y.Z. is a co-founder and significant equity holder of Nuprobe Global, a startup commercializing BDA technology

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Wu, L.R., Chen, S.X., Wu, Y. et al. Multiplexed enrichment of rare DNA variants via sequence-selective and temperature-robust amplification. Nat Biomed Eng 1, 714–723 (2017). https://doi.org/10.1038/s41551-017-0126-5

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