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

BioTechniques

卷:69

Luciano Martelotto¹  Heidi Fettke²  Maria Docanto²  Tu Nguyen-Dumont²  Edmond M Kwan²  Jason A Steen³  Arun A Azad³  Patricia Bukczynska⁴  Christine Hauser⁴  Melissa C Southey⁴  Shivakumar Keerthikumar⁵  David Goode⁵  Nicole Ng⁶ 

[1] Eliza Hall Institute of Medical Research, Melbourne, Australia;

[2] 1Department of Medicine, School of Clinical Sciences, Monash University, Melbourne, Australia;

[3] 2Precision Medicine, School of Clinical Sciences at Monash Health, Melbourne, Australia;

[4] 4Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Australia;

[5] 5Computational Cancer Biology Program, Cancer Research Division, Peter MacCallum Cancer Centre, Melbourne, Australia;

[6] 7Walter &

关键词: cell-free DNA;    cfDNA;    circulating tumor DNA;    ctDNA;    liquid biopsy;    molecular barcoding;   

DOI  :  10.2144/btn-2020-0045

来源: DOAJ

【 摘 要 】

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).

【 授权许可】

Unknown