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

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    Author
    Fettke, H; Steen, JA; Kwan, EM; Bukczynska, P; Keerthikumar, S; Goode, D; Docanto, M; Ng, N; Martelotto, L; Hauser, C; ...
    Date
    2020-08-01
    Source Title
    BioTechniques: the journal of laboratory technology for bioresearch
    Publisher
    FUTURE SCI LTD
    University of Melbourne Author/s
    Kwan, Edmond; Nguyen, Binh Thieu Tu; Goode, David; Keerthikumar, Shivakumar; Southey, Melissa; Azad, Arun
    Affiliation
    Clinical Pathology
    Sir Peter MacCallum Department of Oncology
    Surgery (RMH)
    Metadata
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    Document Type
    Journal Article
    Citations
    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 Access
    URI
    http://hdl.handle.net/11343/252478
    DOI
    10.2144/btn-2020-0045
    Abstract
    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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