Analytical validation of an error-corrected ultra-sensitive ctDNA next-generation sequencing assay

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Fettke, H; Steen, JA; Kwan, EM; Bukczynska, P; Keerthikumar, S; Goode, D; Docanto, M; Ng, N; Martelotto, L; Hauser, C; ...Date
2020-08-01Source Title
BioTechniques: the journal of laboratory technology for bioresearchPublisher
FUTURE SCI LTDUniversity of Melbourne Author/s
Kwan, Edmond; Nguyen, Binh Thieu Tu; Goode, David; Keerthikumar, Shivakumar; Southey, Melissa; Azad, ArunAffiliation
Clinical PathologySir Peter MacCallum Department of Oncology
Surgery (RMH)
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Journal ArticleCitations
Fettke, H., Steen, J. A., Kwan, E. M., Bukczynska, P., Keerthikumar, S., Goode, D., Docanto, M., Ng, N., Martelotto, L., Hauser, C., Southey, M. C., Azad, A. A. & Nguyen-Dumont, T. (2020). Analytical validation of an error-corrected ultra-sensitive ctDNA next-generation sequencing assay. BIOTECHNIQUES, 69 (2), pp.133-140. https://doi.org/10.2144/btn-2020-0045.Access Status
Open AccessAbstract
Plasma circulating tumor DNA (ctDNA) analysis has emerged as a minimally invasive means to perform molecular tumor typing. Here we developed a custom ultra-sensitive ctDNA next-generation sequencing assay using molecular barcoding technology and off-the-shelf reagents combined with bioinformatics tools for enhanced ctDNA analysis. Assay performance was assessed via a spike-in experiment and the technique was applied to analyze 41 plasma samples from men with advanced prostate cancer. Orthogonal validation was performed using a commercial assay. Sensitivity and specificity of 93 and 99.5% were recorded for ultra-rare somatic variants (<1%), with high concordance observed between the in-house and commercial assays. The optimized protocol dramatically improved the efficiency of the assay and enabled the detection of low-frequency somatic variants from plasma cell-free DNA (cfDNA).
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