Ultra-Sensitive Mutation Detection and Genome-Wide DNA Copy Number Reconstruction by Error-Corrected Circulating Tumor DNA Sequencing

摘要: BACKGROUND: Circulating free DNA sequencing (cfDNA-Seq) can portray cancer genome landscapes, but highly sensitive and specific technologies are necessary to accurately detect mutations with often low variant frequencies. METHODS: We developed a customizable hybrid-capture cfDNA-Seq technology using off-the-shelf molecular barcodes novel duplex molecule identification tool for enhanced error correction. RESULTS: Modeling based on cfDNA yields from 58 patients showed that this technology, requiring 25 ng of cfDNA, could be applied >95% metastatic colorectal (mCRC). 32-gene, 163.3-kbp target region detected 100% single-nucleotide variants, 0.15% frequency in spike-in experiments. Molecular barcode correction reduced false-positive mutation calls by 97.5%. In 28 consecutively analyzed mCRC, 80 out 91 previously tumor tissue were called the cfDNA. Call rates similar point indels. identified typical mCRC driver whom biopsy had failed or did not include key genes. Mutations only undetectable matched biopsies included subclonal resistance anti-EGFR antibodies KRAS, parallel evolution multiple PIK3CA 2 cases, TP53 originating clonal hematopoiesis. Furthermore, off-target read analysis allowed simultaneous genome-wide copy number profile reconstruction 20 cases. Copy profiles validated low-coverage whole-genome sequencing. CONCLUSIONS: This error-corrected, ultradeep publicly available bioinformatics tools enables broad insights into genomes evolution. ClinicalTrials.gov Identifier: NCT02112357