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Nat Biotechnol. 2016 May;34(5):547-55. doi: 10.1038/nbt.3520. Epub 2016 Mar 28.

Integrated digital error suppression for improved detection of circulating tumor DNA.

Author information

1
Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, California, USA.
2
Department of Medicine, Division of Oncology, Stanford Cancer Institute, Stanford University, Stanford, California, USA.
3
Department of Radiation Oncology, Stanford University, Stanford, California, USA.
4
Stanford Cancer Institute, Stanford University, Stanford, California, USA.
5
Department of Bioengineering, Stanford University, Stanford, California, USA.
6
Department of Pathology, Stanford University, Stanford, California, USA.
7
Division of Thoracic Surgery, Department of Cardiothoracic Surgery, Stanford School of Medicine, Stanford University, Stanford, California, USA.
8
Division of Hematology, Department of Medicine, Stanford Cancer Institute, Stanford University, Stanford, California, USA.

Abstract

High-throughput sequencing of circulating tumor DNA (ctDNA) promises to facilitate personalized cancer therapy. However, low quantities of cell-free DNA (cfDNA) in the blood and sequencing artifacts currently limit analytical sensitivity. To overcome these limitations, we introduce an approach for integrated digital error suppression (iDES). Our method combines in silico elimination of highly stereotypical background artifacts with a molecular barcoding strategy for the efficient recovery of cfDNA molecules. Individually, these two methods each improve the sensitivity of cancer personalized profiling by deep sequencing (CAPP-Seq) by about threefold, and synergize when combined to yield ∼15-fold improvements. As a result, iDES-enhanced CAPP-Seq facilitates noninvasive variant detection across hundreds of kilobases. Applied to non-small cell lung cancer (NSCLC) patients, our method enabled biopsy-free profiling of EGFR kinase domain mutations with 92% sensitivity and >99.99% specificity at the variant level, and with 90% sensitivity and 96% specificity at the patient level. In addition, our approach allowed monitoring of NSCLC ctDNA down to 4 in 10(5) cfDNA molecules. We anticipate that iDES will aid the noninvasive genotyping and detection of ctDNA in research and clinical settings.

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