Abstract
The sensitivity of conventional DNA sequencing in tumor biopsies is limited by stromal contamination and by genetic heterogeneity within the cancer. Here, we show that microreactor-based pyrosequencing can detect rare cancer-associated sequence variations by independent and parallel sampling of multiple representatives of a given DNA fragment. This technology can thereby facilitate accurate molecular diagnosis of heterogeneous cancer specimens and enable patient selection for targeted cancer therapies. NOTE: In the version of this article initially published, it should have been acknowledged that Jan F. Simons, in addition to Roman K. Thomas and Elizabeth Nickerson, contributed equally to this work. The error has been corrected in the HTML and PDF versions of the article.
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Change history
13 September 2006
In the version of this article initially published, it should have been acknowledged that Jan F. Simons, in addition to Roman K. Thomas and Elizabeth Nickerson, contributed equally to this work. The error has been corrected in the HTML and PDF versions of the article.
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Acknowledgements
This work was supported by the Deutsche Krebshilfe through a Mildred-Scheel Fellowship for Cancer Research to R.K.T. W.R.S. and M.M. are supported by the Novartis Research Foundation, the Claudia Adams Barr Foundation and the Charles A. Dana Human Cancer Genetics Program of the Dana-Farber Cancer Institute, the Poduska Family Foundation, the Damon-Runyon Cancer Research Foundation, Joan's Legacy, the American Cancer Society (RSG-03-240-01-MGO), the Flight Attendant Medical Research Institute and the US National Cancer Institute (R01CA098185). We are greatly indebted to J. Prensner and J. Baldwin for comments and advice.
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Authors and Affiliations
Contributions
R.K.T., E.N., J.F.S., M.E., W.R.S., J.M.R. and M.M. designed the research. R.K.T, E.N., J.F.S., T.T., Y.Y., T.L., J.C.L., K.S., K.O'N., R.D., T.-H.C., K.A.G., H.G., B.D., C.K.L., W.B., P.A., S.K.H., J.H.L., M.T.R. and G.S.T. performed research and analyzed data. P.A.J., H.S., N.L., K.-K.W., A.M.B., E.J.G. and K.H.D. provided samples and patient information. R.K.T., E.N., J.F.S., L.A.G., M.E., W.R.S. and M.M. wrote the manuscript.
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Competing interests
Matthew Meyerson and William R. Sellers have received funding from and served as consultants for Novartis, where Dr. Sellers is now employed.
Matthew Meyerson has received research funding from Genentech.
Jan F. Simons, Brian Desany, Christine K. Lubeski, Stephen K. Hutchinson, J.H. Leamon, Michael T. Ronan, Gregory S. Turenchalk, Michael Egholm and Jonathan M. Rothberg are employees of 454 Life Sciences.
Elisabeth Nickerson was an employee of 454 Life Sciences at the time the research was conducted; she is now an employee of Helicos BioSciences.
Matthew Meyerson, Pasi A. Jänne and William R. Sellers are inventors on a patent application for EGFR mutation detection as a diagnostic tool in cancer.
Supplementary information
Supplementary Fig. 1
Schema of experimental approach for PCR-based exon resequencing. (PDF 77 kb)
Supplementary Fig. 2
Power as a function of number of reads for various concentrations of mutations in the sample. (PDF 37 kb)
Supplementary Fig. 3
Serial dilution of amplicons containing a single-base substitution or a 12-bp deletion into the corresponding wild-type backgrounds, followed by high-coverage sequencing or low-level mutation detection. (PDF 54 kb)
Supplementary Fig. 4
Picotiter plate sequencing of samples S0373 and S0331. (PDF 373 kb)
Supplementary Fig. 5
Oncogenic transformation by patient-derived EGFR mutations and differential sensitivity of Del-4 with and without T790M mutation. (PDF 77 kb)
Supplementary Fig. 6
Validation of mutations detected by picotiter plate sequencing using extensive cloning and sequencing. (PDF 67 kb)
Supplementary Fig. 7
Picotiter plate sequencing of a paraffin-embedded pleural effusion sample obtained before onset of therapy from patient 12.3. (PDF 510 kb)
Supplementary Table 1
Mutations detected by picotiter sequencing. (PDF 8 kb)
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Thomas, R., Nickerson, E., Simons, J. et al. Sensitive mutation detection in heterogeneous cancer specimens by massively parallel picoliter reactor sequencing. Nat Med 12, 852–855 (2006). https://doi.org/10.1038/nm1437
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DOI: https://doi.org/10.1038/nm1437
