Analytical validation of an error-corrected ultra-sensitive ctDNA next-generation sequencing assay
AuthorFettke, H; Steen, JA; Kwan, EM; Bukczynska, P; Keerthikumar, S; Goode, D; Docanto, M; Ng, N; Martelotto, L; Hauser, C; ...
Source TitleBioTechniques: the journal of laboratory technology for bioresearch
PublisherFUTURE SCI LTD
University of Melbourne Author/sKwan, Edmond; Nguyen, Binh Thieu Tu; Goode, David; Keerthikumar, Shivakumar; Southey, Melissa; Azad, Arun
Sir Peter MacCallum Department of Oncology
Document TypeJournal Article
CitationsFettke, 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 StatusOpen Access
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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