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

Heidi Fettke, Jason A. Steen, Edmond M. Kwan, Patricia Bukczynska, Shivakumar Keerthikumar, David Goode, Maria Docanto, Nicole Ng, Luciano Martelotto, Christine Hauser, Melissa C. Southey, Arun A. Azad, Tu Nguyen-Dumont

Research output: Contribution to journalArticleResearchpeer-review

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).

Original languageEnglish
Pages (from-to)133-140
Number of pages8
JournalBioTechniques
Volume69
Issue number2
DOIs
Publication statusPublished - Aug 2020

Keywords

  • cell-free DNA
  • cfDNA
  • circulating tumor DNA
  • ctDNA
  • liquid biopsy
  • molecular barcoding
  • next-generation sequencing

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