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Limitations and opportunities of technologies for the analysis of cell-free DNA in cancer diagnostics

Abstract

Cell-free DNA (cfDNA) in the circulating blood plasma of patients with cancer contains tumour-derived DNA sequences that can serve as biomarkers for guiding therapy, for the monitoring of drug resistance, and for the early detection of cancers. However, the analysis of cfDNA for clinical diagnostic applications remains challenging because of the low concentrations of cfDNA, and because cfDNA is fragmented into short lengths and is susceptible to chemical damage. Barcodes of unique molecular identifiers have been implemented to overcome the intrinsic errors of next-generation sequencing, which is the prevailing method for highly multiplexed cfDNA analysis. However, a number of methodological and pre-analytical factors limit the clinical sensitivity of the cfDNA-based detection of cancers from liquid biopsies. In this Review, we describe the state-of-the-art technologies for cfDNA analysis, with emphasis on multiplexing strategies, and discuss outstanding biological and technical challenges that, if addressed, would substantially improve cancer diagnostics and patient care.

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Fig. 1: cfDNA tests in clinical diagnostics for NSCLC, and treatment workflow.
Fig. 2: Pre-analytical factors impacting the accuracy of cfDNA analysis.
Fig. 3: NGS for cfDNA analysis.
Fig. 4: Primary limitations of NGS-based cfDNA analysis.
Fig. 5: Methods for the accurate detection of mutations with ≤1% in VAF.
Fig. 6: CNVs and gene fusions are challenging biomarkers for cfDNA analysis.
Fig. 7: Accuracy requirements for the screening of early cancers via cfDNA analysis.

References

  1. Lo, Y. D. et al. Rapid clearance of fetal DNA from maternal plasma. Am. J. Hum. Genet. 64, 218–224 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Diehl, F. et al. Circulating mutant DNA to assess tumour dynamics. Nat. Med. 14, 985–990 (2008).

    Article  CAS  PubMed  Google Scholar 

  3. Jahr, S. et al. DNA fragments in the blood plasma of cancer patients: quantitations and evidence for their origin from apoptotic and necrotic cells. Cancer Res. 61, 1659–1665 (2001).

    CAS  PubMed  Google Scholar 

  4. Wan, J. C. et al. Liquid biopsies come of age: towards implementation of circulating tumour DNA. Nat. Rev. Cancer 17, 223–238 (2017).

    Article  CAS  PubMed  Google Scholar 

  5. Diaz, L. A. Jr & Bardelli, A. Liquid biopsies: genotyping circulating tumour DNA. J. Clin. Oncol. 32, 579–586 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  6. Thierry, A. R. et al. Clinical validation of the detection of KRAS and BRAF mutations from circulating tumour DNA. Nat. Med. 20, 430–435 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Alix-Panabieres, C. & Pantel, K. Clinical applications of circulating tumour cells and circulating tumour DNA as liquid biopsy. Cancer Discov. 6, 479–491 (2016).

    Article  CAS  PubMed  Google Scholar 

  8. Mok, T. et al. Detection and dynamic changes of EGFR mutations from circulating tumour DNA as a predictor of survival outcomes in NSCLC patients treated with first-line intercalated erlotinib and chemotherapy. Clin. Cancer Res. 21, 3196–3203 (2015).

    Article  CAS  PubMed  Google Scholar 

  9. Hao, T. B. et al. Circulating cell-free DNA in serum as a biomarker for diagnosis and prognostic prediction of colorectal cancer. Br. J. Cancer 111, 1482–1489 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Azad, A. A. et al. Androgen receptor gene aberrations in circulating cell-free DNA: biomarkers of therapeutic resistance in castration-resistant prostate cancer. Clin. Cancer Res. 21, 2315–2324 (2015).

    Article  CAS  PubMed  Google Scholar 

  11. Lebofsky, R. et al. Circulating tumour DNA as a non-invasive substitute to metastasis biopsy for tumour genotyping and personalized medicine in a prospective trial across all tumour types. Mol. Oncol. 9, 783–790 (2015).

    Article  CAS  PubMed  Google Scholar 

  12. Schwarzenbach, H., Hoon, D. S. B. & Pantel, K. Cell-free nucleic acids as biomarkers in cancer patients. Nat. Rev. Cancer 11, 426–437 (2011).

    Article  CAS  PubMed  Google Scholar 

  13. Goyal, L. et al. Polyclonal secondary FGFR2 mutations drive acquired resistance to FGFR inhibition in patients with FGFR2 fusion-positive cholangiocarcinoma. Cancer Discov. 7, 252–263 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  14. Distribution of In Vitro Diagnostic Products Labeled for Research Use Only or Investigational Use Only: Guidance for Industry and Food and Drug Administration Staff (US FDA, 2013); https://www.fda.gov/regulatory-information/search-fda-guidance-documents/distribution-vitro-diagnostic-productslabeled-research-use-only-or-investigational-use-only

  15. Dietel, M. et al. Diagnostic procedures for non-small-cell lung cancer (NSCLC): recommendations of the European Expert Group. Thorax 71, 177–184 (2015).

    Article  PubMed  Google Scholar 

  16. Herbst, R. S. et al. Pembrolizumab versus docetaxel for previously treated, PD-L1-positive, advanced non-small-cell lung cancer (KEYNOTE-010): a randomised controlled trial. Lancet 387, 1540–1550 (2016).

    Article  CAS  PubMed  Google Scholar 

  17. Vansteenkiste, J. et al. Final results of a multi-center, double-blind, randomized, placebo-controlled phase II study to assess the efficacy of MAGE-A3 immunotherapeutic as adjuvant therapy in stage IB/II non-small cell lung cancer (NSCLC). J. Clin. Oncol. 25, 7554 (2007).

    Article  Google Scholar 

  18. Gettinger, S. N. et al. Overall survival and long-term safety of nivolumab (anti-programmed death 1 antibody, BMS-936558, ONO-4538) in patients with previously treated advanced non-small-cell lung cancer. J. Clin. Oncol. 33, 2004–2012 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Burstein, H. J. et al. Clinical cancer advances 2017: annual report on progress against cancer from the American Society of Clinical Oncology. J. Clin. Oncol. 35, 1341–1367 (2017).

    Article  PubMed  Google Scholar 

  20. Wender, R. et al. American Cancer Society lung cancer screening guidelines. CA Cancer J. Clin. 63, 107–117 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  21. Baldwin, D. R. et al. UK Lung Screen (UKLS) nodule management protocol: modelling of a single screen randomised controlled trial of low-dose CT screening for lung cancer. Thorax 66, 308–313 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Field, J. K. et al. International association for the study of lung cancer computed tomography screening workshop 2011 report. J. Thorac. Oncol. 7, 10–19 (2012).

    Article  PubMed  Google Scholar 

  23. Spigel, D. R. et al. Final efficacy results from OAM4558g, a randomized phase II study evaluating MetMAb or placebo in combination with erlotinib in advanced NSCLC. J. Clin. Oncol. 29, 7505 (2011).

    Article  Google Scholar 

  24. Mok, T. S. et al. Osimertinib or platinum-pemetrexed in EGFR T790M positive lung cancer. N. Engl. J. Med. 376, 629–640 (2017).

    Article  CAS  PubMed  Google Scholar 

  25. Zhou, C. et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer. Lancet Oncol. 12, 735–742 (2011).

    Article  CAS  PubMed  Google Scholar 

  26. Gatzemeier, U. et al. Results of a phase III trial of erlotinib (OSI-774) combined with cisplatin and gemcitabine (GC) chemotherapy in advanced non-small-cell lung cancer (NSCLC). J. Clin. Oncol. 22, 7010 (2004).

    Article  Google Scholar 

  27. Oxnard, G. R. et al. Association between plasma genotyping and outcomes of treatment with osimertinib (AZD9291) in advanced non-small-cell lung cancer. J. Clin. Oncol. 34, 3375–3382 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Clark, T. A. et al. Analytical validation of a hybrid capture-based next-generation sequencing clinical assay for genomic profiling of cell-free circulating tumour DNA. The. J. Mol. Diagnostics 20, 686–702 (2018).

    Article  CAS  Google Scholar 

  29. Lanman, R. B. et al. Analytical and clinical validation of a digital sequencing panel for quantitative, highly accurate evaluation of cell-free circulating tumour DNA. PLoS ONE 10, e0140712 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  30. Paz-Ares, L. et al. plus chemotherapy for squamous non–small-cell lung cancer. N. Engl. J. Med. 379, 2040–2051 (2018).

    Article  CAS  PubMed  Google Scholar 

  31. Fabrizio, D. et al. A blood-based next-generation sequencing assay to determine tumour mutational burden (bTMB) is associated with benefit to an anti-PD-L1 inhibitor, atezolizumab. Cancer Res. 78, 5706–5706 (2018).

    Article  Google Scholar 

  32. Hellmann, M. D. et al. (2018). Nivolumab plus ipilimumab in lung cancer with a high tumour mutational burden. N. Engl. J. Med. 378, 2093–2104 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Gandara, D. R. et al. Blood-based tumour mutational burden as a predictor of clinical benefit in non-small-cell lung cancer patients treated with atezolizumab. Nat. Med. 24, 1441–1448 (2018).

    Article  CAS  PubMed  Google Scholar 

  34. Le, D. T. et al. PD-1 blockade in tumours with mismatch-repair deficiency. N. Engl. J. Med. 372, 2509–2520 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Le, D. T. et al. Mismatch repair deficiency predicts response of solid tumours to PD-1 blockade. Science 357, 409–413 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Rolfo, C. et al. Liquid biopsies in lung cancer: the new ambrosia of researchers. Biochim. Biophys. Acta 1846, 539–546 (2014).

    CAS  PubMed  Google Scholar 

  37. Xiong, L. et al. Dynamics of EGFR mutations in plasma recapitulates the clinical response to EGFR-TKIs in NSCLC patients. Oncotarget 8, 63846–63856 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  38. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Goldberg, S. B. et al. Early assessment of lung cancer immunotherapy response via circulating tumour DNA. Clin. Cancer Res. 24, 1872–1880 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Narayan, A. et al. Ultrasensitive measurement of hotspot mutations in tumour DNA in blood using error-suppressed multiplexed deep sequencing. Cancer Res. 72, 3492–3498 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Thress, K. S. et al. Acquired EGFR C797S mutation mediates resistance to AZD9291 in non-small-cell lung cancer harboring EGFR T790M. Nat. Med. 21, 560–562 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Kwong, L. N. et al. Oncogenic NRAS signaling differentially regulates survival and proliferation in melanoma. Nat. Med. 18, 1503–1510 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Wang, Z. et al. Lung adenocarcinoma harboring EGFR T790M and in trans C797S responds to combination therapy of first-and third-generation EGFR TKIs and shifts allelic configuration at resistance. J. Thorac. Oncol. 12, 1723–1727 (2017).

    Article  PubMed  Google Scholar 

  44. Corcoran, R. B. & Chabner, B. A. Application of cell-free DNA analysis to cancer treatment. N. Engl. J. Med. 379, 1754–1765 (2018).

    Article  CAS  PubMed  Google Scholar 

  45. Garcia-Murillas, I. et al. Mutation tracking in circulating tumour DNA predicts relapse in early breast cancer. Sci. Transl. Med. 7, 302ra133 (2015).

    Article  PubMed  Google Scholar 

  46. Dawson, S. J. et al. Analysis of circulating tumour DNA to monitor metastatic breast cancer. N. Engl. J. Med. 368, 1199–1209 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Reinert, T. et al. Analysis of plasma cell-free DNA by ultradeep sequencing in patients with stages I to III colorectal cancer. JAMA Oncol. 5, 1124–1131 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Tie, J. et al. Circulating tumour 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 

  49. Abbosh, C., Birkbak, N. J. & Swanton, C. Early stage NSCLC—challenges to implementing ctDNA-based screening and MRD detection. Nat. Rev. Clin. Oncol. 15, 577–586 (2018).

    Article  CAS  PubMed  Google Scholar 

  50. Oxnard, G. R. et al. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clin. Cancer Res. 20, 1698–1705 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Phallen, J. et al. Direct detection of early-stage cancers using circulating tumour DNA. Sci. Transl. Med. 9, eaan2415 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  52. Imperiale, T. F. et al. Multitarget stool DNA testing for colorectal-cancer screening. N. Engl. J. Med. 370, 1287–1297 (2014).

    Article  CAS  PubMed  Google Scholar 

  53. Chan, K. A. et al. Analysis of plasma Epstein-Barr virus DNA to screen for nasopharyngeal cancer. N. Engl. J. Med. 377, 513–522 (2017).

    Article  CAS  PubMed  Google Scholar 

  54. Cohen, J. D. et al. Detection and localization of surgically resectable cancers with a multi-analyte blood test. Science 359, 926–930 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Lecomte, T. et al. Detection of free circulating tumour associated DNA in plasma of colorectal cancer patients and its association with prognosis. N. Engl. J. Med. 370, 1287–1297 (2014).

    Google Scholar 

  56. Hu, Y. et al. False-positive plasma genotyping due to clonal hematopoiesis. Clin. Cancer Res. 24, 4437–4443 (2018).

    Article  CAS  PubMed  Google Scholar 

  57. Heitzer, E., Haque, I. S., Roberts, C. E. & Speicher, M. R. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat. Rev. Genet. 20, 71–88 (2019).

    Article  CAS  PubMed  Google Scholar 

  58. Sina, A. A. I. et al. Epigenetically reprogrammed methylation landscape drives the DNA self-assembly and serves as a universal cancer biomarker. Nat. Commun. 9, 4915 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  59. Lehmann-Werman, R. et al. Identification of tissue-specific cell death using methylation patterns of circulating DNA. Proc. Natl Acad. Sci. USA 113, E1826–E1834 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Deng, J. et al. Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming. Nat. Biotechnol. 27, 353–360 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Xu, R. H. et al. Circulating tumour DNA methylation markers for diagnosis and prognosis of hepatocellular carcinoma. Nat. Mater. 16, 1155–1161 (2017).

    Article  CAS  PubMed  Google Scholar 

  62. Teschendorff, A. E. & Relton, C. L. Statistical and integrative system-level analysis of DNA methylation data. Nat. Rev. Genet. 19, 129–147 (2018).

    Article  CAS  PubMed  Google Scholar 

  63. Guo, S. et al. Identification of methylation haplotype blocks aids in deconvolution of heterogeneous tissue samples and tumour tissue-of-origin mapping from plasma DNA. Nat. Genet. 49, 635–642 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Tug, S. et al. Exercise-induced increases in cell-free DNA in human plasma originate predominantly from cells of the haematopoietic lineage. Exerc. Immunol. Rev. 21, 164–173 (2015).

    PubMed  Google Scholar 

  65. Moreira, V. G., Prieto, B., Rodr.ıguez, J. S. M. & Alvarez, F. V. Usefulness of cell-free plasma DNA, procalcitonin and C-reactive protein as markers of infection in febrile patients. Ann. Clin. Biochem. 47, 253–258 (2010).

    Article  CAS  PubMed  Google Scholar 

  66. Burnham, P. et al. Urinary cell-free DNA is a versatile analyte for monitoring infections of the urinary tract. Nat. Commun. 9, 2412 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  67. Siljan, W. W. et al. Circulating cell-free DNA is elevated in community acquired bacterial pneumonia and predicts short-term outcome. J. Infect. 73, 383–386 (2016).

    Article  PubMed  Google Scholar 

  68. Lindahl, T. Instability and decay of the primary structure of DNA. Nature 362, 709–715 (1993).

    Article  CAS  PubMed  Google Scholar 

  69. Richter, C., Park, J. W. & Ames, B. N. Normal oxidative damage to mitochondrial and nuclear DNA is extensive. Proc. Natl Acad. Sci. USA 85, 6465–6467 (1988).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  70. Cooke, M. S., Evans, M. D., Dizdaroglu, M. & Lunec, J. Oxidative DNA damage: mechanisms, mutation, and disease. FASEB J. 17, 1195–1214 (2003).

    Article  CAS  PubMed  Google Scholar 

  71. Norton, S. E., Lechner, J. M., Williams, T. & Fernando, M. R. A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood sample storage and shipping as determined by digital PCR. Clin. Biochem. 46, 1561–1565 (2013).

    Article  CAS  PubMed  Google Scholar 

  72. Manning, J. E. in Emergency Medicine: A Comprehensive Study Guide (ed. Tintinalli, J. E.) 227 (McGraw-Hill, 2004).

  73. Botezatu, I. et al. Genetic analysis of DNA excreted in urine: a new approach for detecting specific genomic DNA sequences from cells dying in an organism. Clin. Chem. 46, 1078–1084 (2000).

    Article  CAS  PubMed  Google Scholar 

  74. Koide, K. et al. Fragmentation of cell-free fetal DNA in plasma and urine of pregnant women. Prenat. Diagnosis 25, 604–607 (2005).

    Article  CAS  Google Scholar 

  75. Tani, M. & Beck, S. Epigenome-wide association studies for cancer biomarker discovery in circulating cell-free DNA: technical advances and challenges. Curr. Opin. Genet. Dev. 42, 48–55 (2017).

    Article  Google Scholar 

  76. Reckamp, K. L. et al. A highly sensitive and quantitative test platform for detection of NSCLC EGFR mutations in urine and plasma. J. Thorac. Oncol. 11, 1690–1700 (2016).

    Article  PubMed  Google Scholar 

  77. Swanson, P. et al. Performance of the automated Abbott RealTime HIV-1 assay on a genetically diverse panel of specimens from London: comparison to VERSANT HIV-1 RNA 3.0, AMPLICOR HIV-1 MONITOR v1. 5, and LCx^A R HIV RNA quantitative assays. J. Virol. Methods 137, 184–192 (2006).

    Article  CAS  PubMed  Google Scholar 

  78. Castle, P. E. Performance of carcinogenic human papillomavirus (HPV) testing and HPV16 or HPV18 genotyping for cervical cancer screening of women aged 25 years and older: a subanalysis of the ATHENA study. Lancet Oncol. 12, 880–890 (2011).

    Article  PubMed  Google Scholar 

  79. Sandri, M. T. et al. Comparison of the Digene HC2 assay and the Roche AMPLICOR human papillomavirus (HPV) test for detection of high-risk HPV genotypes in cervical samples. J. Clin. Microbiol. 44, 2141–2146 (2006).

    Article  PubMed  PubMed Central  Google Scholar 

  80. Misawa, Y. et al. Application of loop-mediated isothermal amplification technique to rapid and direct detection of methicillin-resistant Staphylococcus aureus (MRSA) in blood cultures. J. Infect. Chemother. 13, 134–140 (2007).

    Article  CAS  PubMed  Google Scholar 

  81. Ethridge, S. F. et al. Performance of the Aptima HIV-1 RNA qualitative assay with 16- and 32-member specimen pools. J. Clin. Microbiol. 48, 3343–3345 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  82. Pao, W. et al. Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain. PLoS Med. 2, e73 (2005).

    Article  PubMed  PubMed Central  Google Scholar 

  83. Mok, T. S. et al. Osimertinib or platinum–pemetrexed in EGFR T790M–positive lung cancer. N. Engl. J. Med. 376, 629–640 (2017).

    Article  CAS  PubMed  Google Scholar 

  84. Hindson, B. J. et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83, 8604–8610 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Newton, C. R. et al. Analysis of any point mutation in DNA. The amplification refractory mutation system (ARMS). Nucleic Acids Res. 17, 2503–2516 (1989).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Das, J. et al. An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum. Nat. Chem. 7, 569–575 (2015).

    Article  CAS  PubMed  Google Scholar 

  87. Lin, M. et al. Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform. Nat. Protoc. 11, 1244–1263 (2016).

    Article  CAS  PubMed  Google Scholar 

  88. Gootenberg, J. S. et al. Nucleic acid detection with CRISPR-Cas13a/C2c2. Science 356, 438–442 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Dressman, D., Yan, H., Traverso, G., Kinzler, K. W. & Vogelstein, B. Transforming single DNA molecules into fluorescent magnetic particles for detection and enumeration of genetic variations. Proc. Natl Acad. Sci. USA 100, 8817–8822 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Khoo, C. et al. Molecular methods for somatic mutation testing in lung adenocarcinoma: EGFR and beyond. Transl. Lung Cancer Res. 4, 126–141 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Ragoussis, J. Genotyping technologies for genetic research. Annu. Rev. Genomics Hum. Genet. 10, 117–133 (2009).

    Article  CAS  PubMed  Google Scholar 

  92. Murtaza, M. et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 497, 108–112 (2013).

    Article  CAS  PubMed  Google Scholar 

  93. Wetterstrand, K. A. DNA Sequencing Costs: Data from the NHGRI Genome Sequencing Program (GSP) (National Human Genome Research Institute, accessed 19 July 2018); www.genome.gov/sequencingcostsdata

  94. Laver, T. et al. Assessing the performance of the Oxford Nanopore Technologies MinION. Biomol. Detect. Quantif. 3, 1–8 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  95. Carneiro, M. O. et al. Pacific biosciences sequencing technology for genotyping and variation discovery in human data. BMC Genomics 13, 375 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Taylor, A. D., Micheel, C. M., Anderson, I. A., Levy, M. A. & Lovly, C. M. The path (way) less traveled: a pathway-oriented approach to providing information about precision cancer medicine on My Cancer Genome. Transl. Oncol. 9, 163–165 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  97. Diehn, M. et al. Early prediction of clinical outcomes in resected stage II and III colorectal cancer (CRC) through deep sequencing of circulating tumour DNA (ctDNA). J. Clin. Oncol. 35, 3591 (2017).

    Article  Google Scholar 

  98. Couraud, S. et al. Non-invasive diagnosis of actionable mutations by deep sequencing of circulating-free DNA in non-small-cell lung cancer: findings from BioCAST/IFCT-1002. Clin. Cancer Res. 20, 4613–4624 (2014).

    Article  CAS  PubMed  Google Scholar 

  99. Song, P. 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).

    Article  CAS  PubMed  Google Scholar 

  100. Meyer, M. & Kircher, M. Illumina sequencing library preparation for highly multiplexed target capture and sequencing. Cold Spring Harb. Protoc. 2010, pdb.prot5448 (2010).

    Article  PubMed  Google Scholar 

  101. Hovelson, D. H. et al. Development and validation of a scalable next-generation sequencing system for assessing relevant somatic variants in solid tumours. Neoplasia 17, 385–399 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Dupuis, J. R. et al. HiMAP: robust phylogenomics from highly multiplexed amplicon sequencing. Mol. Ecol. Resour. 18, 1000–1019 (2018).

    Article  CAS  Google Scholar 

  103. 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 

  104. Blakely, C. M. et al. Evolution and clinical impact of co-occurring genetic alterations in advanced-stage EGFR-mutant lung cancers. Nat. Genet. 49, 1693–1704 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Kinde, I. et al. Detection and quantification of rare mutations with massively parallel sequencing. Proc. Natl Acad. Sci. USA 108, 9530–9535 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  106. Lo, Y. D. et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci. Transl. Med. 2, 61ra91 (2010).

    Article  CAS  PubMed  Google Scholar 

  107. Thierry, A. R. et al. Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts. Nucleic Acids Res. 38, 6159–6175 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Snyder, M. W., Kircher, M., Hill, A. J., Daza, R. M. & Shendure, J. Cell-free DNA comprises an in vivo nucleosome footprint that informs its tissues-of-origin. Cell 164, 57–68 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Mouliere, F. et al. Enhanced detection of circulating tumour DNA by fragment size analysis. Sci. Transl. Med. 10, 466 (2018).

    Article  Google Scholar 

  110. Thakur, B. K. et al. Double-stranded DNA in exosomes: a novel biomarker in cancer detection. Cell Res. 24, 766–769 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Balaj, L. et al. Tumour microvesicles contain retrotransposon elements and amplified oncogene sequences. Nat. Commun. 2, 180 (2011).

    Article  PubMed  Google Scholar 

  112. Zhang, J. X. et al. Predicting DNA hybridization kinetics from sequence. Nat. Chem. 10, 91–98 (2018).

    Article  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Kou, R. et al. Benefits and challenges with applying unique molecular identifiers in next generation sequencing to detect low frequency mutations. PLoS ONE 11, e0146638 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  115. Jabara, C. B., Jones, C. D., Roach, J., Anderson, J. A. & Swanstrom, R. Accurate sampling and deep sequencing of the HIV-1 protease gene using a Primer ID. Proc. Natl Acad. Sci. USA 108, 20166–20171 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Abascal, F. et al. Somatic mutation landscapes at single-molecule resolution. Nature 593, 405–410 (2021).

    Article  CAS  PubMed  Google Scholar 

  117. Cohen, J. D. et al. Detection of low-frequency DNA variants by targeted sequencing of the Watson and Crick strands. Nat. Biotechnol. 39, 1220–1227 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Pel, J. et al. Nonlinear electrophoretic response yields a unique parameter for separation of biomolecules. Proc. Natl Acad. Sci. USA 106, 14796–14801 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Kidess, E. et al. Mutation profiling of tumour DNA from plasma and tumour tissue of colorectal cancer patients with a novel, high-sensitivity multiplexed mutation detection platform. Oncotarget 6, 2549–2561 (2015).

    Article  PubMed  Google Scholar 

  120. Song, C. & Liu 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 

  121. Seyama, T. et al. A novel blocker-PCR method for detection of rare mutant alleles in the presence of an excess amount of normal DNA. Nucleic Acids Res. 20, 2493–2496 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Arcila, M., Lau, C., Nafa, K. & Ladanyi, M. Detection of KRAS and BRAF mutations in colorectal carcinoma: roles for high-sensitivity locked nucleic acid-PCR sequencing and broad-spectrum mass spectrometry genotyping. J. Mol. Diagnostics 13, 64–73 (2011).

    Article  CAS  Google Scholar 

  123. Orum, H. et al. Single base pair mutation analysis by PNA directed PCR clamping. Nucleic Acids Res. 21, 5332–5336 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. 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 (2011).

    Article  PubMed  Google Scholar 

  125. 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 

  126. Zuo, Z. et al. Application of COLD-PCR for improved detection of KRAS mutations in clinical samples. Mod. Pathol. 22, 1023–1031 (2009).

    Article  CAS  PubMed  Google Scholar 

  127. Wu, L. R. et al. 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 

  128. Ciriello, G. et al. Emerging landscape of oncogenic signatures across human cancers. Nat. Genet. 45, 1127–1133 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  129. Mertens, F., Johansson, B., Fioretos, T. & Mitelman, F. The emerging complexity of gene fusions in cancer. Nat. Rev. Cancer 15, 371–381 (2015).

    Article  CAS  PubMed  Google Scholar 

  130. Wang, Q. et al. Application of next generation sequencing to human gene fusion detection: computational tools, features and perspectives. Brief. Bioinform. 14, 506–519 (2012).

    Article  PubMed  PubMed Central  Google Scholar 

  131. Maher, C. A. et al. Transcriptome sequencing to detect gene fusions in cancer. Nature 458, 97–101 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  132. Zhao, Q. et al. Transcriptome-guided characterization of genomic rearrangements in a breast cancer cell line. Proc. Natl Acad. Sci. USA 106, 1886–1891 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Zack, T. I. et al. Pan-cancer patterns of somatic copy number alteration. Nat. Genet. 45, 1134–1140 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  134. Whale, A. S. et al. Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic Acids Res. 40, e82 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  135. Pinkel, D. et al. High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays. Nat. Genet. 20, 207–211 (1998).

    Article  CAS  PubMed  Google Scholar 

  136. Chiu, R. W. et al. Non-invasive prenatal assessment of trisomy 21 by multiplexed maternal plasma DNA sequencing: large scale validity study. Brit. Med. J. 342, c7401 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  137. Kops, G. J., Weaver, B. A. & Cleveland, D. W. On the road to cancer: aneuploidy and the mitotic checkpoint. Nat. Rev. Cancer 5, 773–785 (2004).

    Article  Google Scholar 

  138. Mandel, P. & Metais, P. Les acides nucleiques du plasma sanguin chez la homme. C. R. Seances Soc. Biol. Fil. 142, 241–243 (1948).

    CAS  PubMed  Google Scholar 

  139. Bettegowda, C. et al. Detection of circulating tumour DNA in early- and late-stage human malignancies. Sci. Transl. Med. 6, 224ra224 (2014).

    Article  Google Scholar 

  140. Aravanis, A. M., Lee, M. & Klausner, R. D. Next-generation sequencing of circulating tumour DNA for early cancer detection. Cell 168, 571–574 (2017).

    Article  CAS  PubMed  Google Scholar 

  141. Razavi, P. et al. Cell-free DNA (cfDNA) mutations from clonal hematopoiesis: implications for interpretation of liquid biopsy tests. J. Clin. Oncol. 35, 11526 (2017).

    Article  Google Scholar 

  142. Genovese, G. et al. Clonal hematopoiesis and blood-cancer risk inferred from blood DNA sequence. N. Engl. J. Med. 371, 2477–2487 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  143. Schwarzenbach, H., Nishida, N., Calin, G. A. & Pantel, K. Clinical relevance of circulating cell-free microRNAs in cancer. Nat. Rev. Clin. Oncol. 11, 145–156 (2014).

    Article  CAS  PubMed  Google Scholar 

  144. Azmi, A. S., Bao, B. & Sarkar, F. H. Exosomes in cancer development, metastasis, and drug resistance: a comprehensive review. Cancer Metastasis Rev. 32, 623–642 (2013).

    Article  CAS  PubMed  Google Scholar 

  145. De Mattos-Arruda, L. et al. Circulating tumour cells and cell-free DNA as tools for managing breast cancer. Nat. Rev. Clin. Oncol. 10, 377–389 (2013).

    Article  PubMed  Google Scholar 

  146. de Bono, J. S. et al. Circulating tumour cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin. Cancer Res. 14, 6302–6309 (2008).

    Article  CAS  PubMed  Google Scholar 

  147. Potapov, V. & Ong, J. L. Examining sources of error in PCR by single-molecule sequencing. PLoS ONE 12, e0181128 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  148. Schirmer, M. et al. Insight into biases and sequencing errors for amplicon sequencing with the Illumina MiSeq platform. Nucleic Acids Res. 43, e37 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  149. Schirmer, M., Damore, R., Ijaz, U. Z., Hall, N. & Quince, C. Illumina error profiles: resolving fine-scale variation in metagenomic sequencing data. BMC Bioinformatics 17, 125 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  150. Minoche, A. E., Dohm, J. C. & Himmelbauer, H. Evaluation of genomic high-throughput sequencing data generated on Illumina HiSeq and genome analyzer systems. Genome Biol. 12, R112 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Jain, M. et al. Nanopore sequencing and assembly of a human genome with ultra-long reads. Nat. Biotechnol. 36, 338–345 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Quail, M. A. et al. A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers. BMC Genomics 13, 341 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Leary, R. J. et al. Development of personalized tumour biomarkers using massively parallel sequencing. Sci. Transl. Med. 2, 20ra14 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank S. Skates for useful discussions regarding diagnostic economics and outcomes. L.N.K. was supported by NIH grant P01CA163222. A.A.P. was supported by NIH grants R01CA197486 and R01CA233364. D.Y.Z. was supported by NIH grants R01CA203964 and R01CA233364, and by CPRIT grant RP180147.

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P.S., L.R.W. and D.Y.Z. wrote the paper on the basis of in-depth discussions with all authors.

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Correspondence to David Yu Zhang.

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P.S., L.R.W. and A.A.P. are consultants for NuProbe USA. A.A.P. is a consultant for Binary Genomics and has equity ownership in the company. D.Y.Z. is a co-founder of, and holds significant equity in, NuProbe Global, Torus Biosystems and Pana Bio.

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Song, P., Wu, L.R., Yan, Y.H. et al. Limitations and opportunities of technologies for the analysis of cell-free DNA in cancer diagnostics. Nat. Biomed. Eng 6, 232–245 (2022). https://doi.org/10.1038/s41551-021-00837-3

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