TY - JOUR
T1 - Development and Application of Duplex Sequencing Strategy for Cell-Free DNA–Based Longitudinal Monitoring of Stage IV Colorectal Cancer
AU - Mallampati, Saradhi
AU - Zalles, Stephanie
AU - Duose, Dzifa Y.
AU - Hu, Peter C.
AU - Medeiros, L. Jeffrey
AU - Wistuba, Ignacio I.
AU - Kopetz, Scott
AU - Luthra, Rajyalakshmi
N1 - Publisher Copyright:
© 2019 American Society for Investigative Pathology and the Association for Molecular Pathology
PY - 2019/11
Y1 - 2019/11
N2 - Potential applications of cell-free DNA (cfDNA)–based molecular profiling have used in patients with diverse malignant tumors. However, capturing all cfDNA that originates from tumor cells and identifying true variants present in this minute fraction remain challenges to the widespread application of cfDNA-based liquid biopsies in the clinical setting. In this study, we evaluate a systematic approach and identify key components of wet bench and bioinformatics strategies to address these challenges. We found that concentration of enrichment oligonucleotides, elements of the library preparation, and the structure of adaptors are critical for achieving high enrichment of target regions, retaining variant allele frequencies accurately throughout all involved steps of library preparation, and obtaining high variant coverage. We developed a dual molecular barcode–integrated error elimination strategy to remove sequencing artifacts and a background error correction strategy to distinguish true variants from abundant false-positive variants. We further describe a clinical application of this cfDNA-based duplex sequencing approach that can be used to monitor disease progression in patients with stage IV colorectal cancer. The findings also suggest that cfDNA-based molecular testing observations are highly concordant with observations obtained by traditional imaging methods. Overall, the findings presented in this study have potential implications for early detection of cancer, identification of minimal residual disease, and evaluation of therapeutic responses in patients with cancer.
AB - Potential applications of cell-free DNA (cfDNA)–based molecular profiling have used in patients with diverse malignant tumors. However, capturing all cfDNA that originates from tumor cells and identifying true variants present in this minute fraction remain challenges to the widespread application of cfDNA-based liquid biopsies in the clinical setting. In this study, we evaluate a systematic approach and identify key components of wet bench and bioinformatics strategies to address these challenges. We found that concentration of enrichment oligonucleotides, elements of the library preparation, and the structure of adaptors are critical for achieving high enrichment of target regions, retaining variant allele frequencies accurately throughout all involved steps of library preparation, and obtaining high variant coverage. We developed a dual molecular barcode–integrated error elimination strategy to remove sequencing artifacts and a background error correction strategy to distinguish true variants from abundant false-positive variants. We further describe a clinical application of this cfDNA-based duplex sequencing approach that can be used to monitor disease progression in patients with stage IV colorectal cancer. The findings also suggest that cfDNA-based molecular testing observations are highly concordant with observations obtained by traditional imaging methods. Overall, the findings presented in this study have potential implications for early detection of cancer, identification of minimal residual disease, and evaluation of therapeutic responses in patients with cancer.
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U2 - 10.1016/j.jmoldx.2019.06.008
DO - 10.1016/j.jmoldx.2019.06.008
M3 - Article
C2 - 31401123
AN - SCOPUS:85073327768
SN - 1525-1578
VL - 21
SP - 994
EP - 1009
JO - Journal of Molecular Diagnostics
JF - Journal of Molecular Diagnostics
IS - 6
ER -