DNA sequence variants with allele fractions below 1% are difficult to detect and quantify by sequencing owing to intrinsic errors in sequencing-by-synthesis methods. Although molecular-identifier barcodes can detect mutations with a variant-allele frequency (VAF) as low as 0.1% using next-generation sequencing (NGS), sequencing depths of over 25,000× are required, thus hampering the detection of mutations at high sensitivity in patient samples and in most samples used in research. Here we show that low-frequency DNA variants can be detected via low-depth multiplexed NGS after their amplification, by a median of 300-fold, using polymerase chain reaction and rationally designed ‘blocker’ oligonucleotides that bind to the variants. Using an 80-plex NGS panel and a sequencing depth of 250×, we detected single nucleotide polymorphisms with a VAF of 0.019% and contamination in human cell lines at a VAF as low as 0.07%. With a 16-plex NGS panel covering 145 mutations across 9 genes involved in melanoma, we detected low-VAF mutations (0.2–5%) in 7 out of the 19 samples of freshly frozen tumour biopsies, suggesting that tumour heterogeneity could be notably higher than previously recognized.
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The main data supporting the results in this study are available within the paper and its Supplementary Information. All requests for raw and analysed data will be reviewed by the Legal Department of Rice University to verify whether the request is subject to any intellectual property or confidentiality constraints. Requests for patient-related data not included in the paper will not be considered. Data can be shared for non-commercial research purposes via a material transfer agreement.
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Goodwin, S., McPherson, J. D. & McCombie, W. R. Coming of age: ten years of next-generation sequencing technologies. Nat. Rev. Genet. 17, 333–351 (2016).
Mardis, E. R. Next-generation sequencing platforms. Annu. Rev. Anal. Chem. 6, 287–303 (2013).
Schirmer, M. et al. Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform. Nucleic Acids Res. 43, e37 (2015).
Schirmer, M., DaAmore, R., Ijaz, U. Z., Hall, N. & Quince, C. Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data. BMC Bioinform. 17, 125 (2016).
Kinde, I., Wu, J., Papadopoulos, N., Kinzler, K. W. & Vogelstein, B. Detection and quantification of rare mutations with massively parallel sequencing. Proc. Natl Acad. Sci. USA 108, 9530–9535 (2011).
Newman, A. M. et al. Integrated digital error suppression for improved detection of circulating tumor DNA. Nat. Biotechnol. 34, 547–555 (2016).
Schmitt, M. W. et al. Detection of ultra-rare mutations by next-generation sequencing. Proc. Natl Acad. Sci. USA 109, 14508–14513 (2012).
Schwaederle, M. C. et al. Utility of genomic assessment of blood-derived circulating tumor DNA (ctDNA) in patients with advanced lung adenocarcinoma. Clin. Cancer Res. 23, 5101–5111 (2017).
Ma, C. X. et al. Neratinib efficacy and circulating tumor DNA detection of HER2 mutations in HER2 nonamplified metastatic breast cancer. Clin. Cancer Res. 23, 5687–5695 (2017).
Hata, A. N. et al. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat. Med. 22, 262–269 (2016).
Cohen, J. D. et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 359, 926–930 (2018).
Zitvogel, L., Ma, Y., Raoult, D., Kroemer, G. & Gajewski, T. F. The microbiome in cancer immunotherapy: diagnostic tools and therapeutic strategies. Science 359, 1366–1370 (2018).
Zhernakova, A. et al. Population-based metagenomics analysis reveals markers for gut microbiome composition and diversity. Science 352, 565–569 (2016).
Fisher, R. A., Gollan, B. & Helaine, S. Persistent bacterial infections and persister cells. Nat. Rev. Microbiol. 15, 453–464 (2017).
Merker, M. et al. Whole genome sequencing reveals complex evolution patterns of multidrug-resistant Mycobacterium tuberculosis Beijing strains in patients. PLoS ONE 8, e82551 (2013).
Hindson, B. J. et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83, 8604–8610 (2011).
Watanabe, M. et al. Ultra-sensitive detection of the pretreatment EGFR T790M mutation in non-small cell lung cancer patients with an EGFR-activating mutation using droplet digital PCR. Clin. Cancer Res. 21, 3552–3560 (2015).
Didelot, A. et al. Competitive allele specific TaqMan PCR for KRAS, BRAF and EGFR mutation detection in clinical formalin fixed paraffin embedded samples. Exp. Mol. Pathol. 92, 275–280 (2012).
Milbury, C. A., Li, J. & Makrigiorgos, G. M. Ice-COLD-PCR enables rapid amplification and robust enrichment for low-abundance unknown DNA mutations. Nucleic Acids Res. 39, e2 (2010).
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).
International HapMap Project (National Human Genome Research Institute, 2012); https://www.genome.gov/10001688/international-hapmap-project/
1000 Genomes Project (The International Genome Sample Resource, 2021); http://www.internationalgenome.org/
Potapov, V. & Ong, J. L. Examining sources of error in PCR by single-molecule sequencing. PLoS ONE 12, e0169774 (2017).
Volokhov, D. V., Graham, L. J., Brorson, K. A. & Chizhikov, V. E. Mycoplasma testing of cell substrates and biologics: review of alternative non-microbiological techniques. Mol. Cell. Probes. 25, 69–77 (2011).
Capes-Davis, A. et al. Check your cultures! A list of cross-contaminated or misidentified cell lines. Int. J. Cancer 127, 1–8 (2010).
Qu, K. et al. Detection of BRAF V600 mutations in metastatic melanoma: comparison of the Cobas 4800 and Sanger sequencing assays. J. Mol. Diagn. 15, 790–795 (2013).
Zehir, A. et al. Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients. Nat. Med. 23, 703–713 (2017).
Beltran, H. et al. Targeted next-generation sequencing of advanced prostate cancer identifies potential therapeutic targets and disease heterogeneity. Eur. Urol. 63, 920–926 (2013).
Russo, M. et al. Tumor heterogeneity and lesion-specific response to targeted therapy in colorectal cancer. Cancer Discov. 6, 147–153 (2016).
McGranahan, N. & Swanton, C. Clonal heterogeneity and tumor evolution: past, present, and the future. Cell 168, 613–628 (2017).
Dagogo-Jack, I. & Shaw, A. T. Tumour heterogeneity and resistance to cancer therapies. Nat. Rev. Clin. Oncol. 15, 81–94 (2018).
Romano, G. et al. A preexisting rare PIK3CAE545K subpopulation confers clinical resistance to MEK plus CDK4/6 inhibition in NRAS melanoma and is dependent on S6K1 signaling. Cancer Discov. 8, 556–567 (2018).
Tate, J. G. et al. COSMIC: the catalogue of somatic mutations in cancer. Nucleic Acids Res. 47, D941–D947 (2018).
Pel, J. et al. Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules. Proc. Natl Acad. Sci. USA 106, 14796–14801 (2009).
Kang, S. et al. Targeted sequencing with enrichment PCR: a novel diagnostic method for the detection of EGFR mutations. Oncotarget 6, 13742–13749 (2015).
Song, C. et al. Elimination of unaltered DNA in mixed clinical samples via nuclease-assisted minor-allele enrichment. Nucleic Acids Res. 44, e146 (2016).
Lee, S. H. et al. CUT-PCR: CRISPR-mediated, ultrasensitive detection of target DNA using PCR. Oncogene 36, 6823–6829 (2017).
Alexandrov, L. B. et al. The repertoire of mutational signatures in human cancer. Nature 578, 94–101 (2020).
Barretina, J. et al. The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity. Nature 483, 603–607 (2012).
Sharma, S. V., Haber, D. A. & Settleman, J. Cell line-based platforms to evaluate the therapeutic efficacy of candidate anticancer agents. Nat. Rev. Cancer 10, 241–253 (2010).
Kikuchi, T. et al. Human iPS cell-derived dopaminergic neurons function in a primate Parkinson’s disease model. Nature 548, 592–596 (2017).
Schweppe, R. E. et al. Deoxyribonucleic acid profiling analysis of 40 human thyroid cancer cell lines reveals cross-contamination resulting in cell line redundancy and misidentification. J. Clin. Endocrinol. Metab. 93, 4331–4341 (2008).
Freedman, L. P. et al. Reproducibility: changing the policies and culture of cell line authentication. Nat. Methods 12, 493–497 (2015).
Huang, Y., Liu, Y., Zheng, C. & Shen, C. Investigation of cross-contamination and misidentification of 278 widely used tumor cell lines. PLoS ONE 12, e0170384 (2017).
Horbach, S. P. & Halffman, W. The ghosts of HeLa: how cell line misidentification contaminates the scientific literature. PLoS ONE 12, e0186281 (2017).
Martincorena, I. et al. High burden and pervasive positive selection of somatic mutations in normal human skin. Science 348, 880–886 (2015).
Do, H. & Dobrovic, A. Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin. Chem. 61, 64–71 (2015).
Chen, G., Mosier, S., Gocke, C. D., Lin, M. T. & Eshleman, J. R. Cytosine deamination is a major cause of baseline noise in next-generation sequencing. Mol. Diagn. Ther. 18, 587–593 (2014).
Goldberg, S. B. et al. Early assessment of lung cancer immunotherapy response via circulating tumor DNA. Clin. Cancer Res. 24, 1872–1880 (2018).
This work was supported by NIH grant nos R01CA203964 and R01CA233364, and CPRIT grant no. RP180147 to D.Y.Z. We thank J. Nie for proofreading assistance and G. Bao for providing access to his BioRad QX200 digital droplet PCR instrument. We thank Nuprobe for providing early access VarMap NSCLC kits for cfDNA testing.
There are patents pending on the BDA (patent number EP3146080B1) and mBDA (patent number WO2019164885A1) methods used in this work. P.S., S.X.C., L.Y.C. and P.D. declare competing interests in the form of consulting for Nuprobe USA. A.A.P. declares a competing interest in the form of consulting for Nuprobe USA as well as consulting for and equity ownership in Binary Genomics. D.Y.Z. declares a competing interest in the form of consulting for and equity ownership in Nuprobe and Torus Biosystems as well as consulting for Avenge Bio.
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Song, P., Chen, S.X., Yan, Y.H. et al. Selective multiplexed enrichment for the detection and quantitation of low-fraction DNA variants via low-depth sequencing. Nat Biomed Eng 5, 690–701 (2021). https://doi.org/10.1038/s41551-021-00713-0
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