Development and Technical Validation of the Clinical Lymphoma Exploration and Research Sequencing (CLEARS) Panel and Its Optimization for ctDNA Analysis.

Abstract

Circulating tumour DNA (ctDNA) is a potential biomarker for the assessment of prognosis and treatment response in lymphoma patients. However, there is still a lack of standardization of ctDNA analysis. Here, we present the development of our Clinical Lymphoma Exploration And Research Sequencing (CLEARS) panel for cancer personalized profiling by deep sequencing (CAPP-Seq), together with optimization of sequencing library preparation for ctDNA analysis. The CLEARS panel targets coding sequences of 521 genes most frequently altered in lymphomas, with its main purpose to analyse ctDNA. We tested its performance, determined the optimal workflow for cell‑free DNA (cfDNA) pre-capture sample library preparation, and confirmed our ability to achieve sufficient variant allele frequency detection limit for baseline ctDNA evaluation. Reported technical considerations and optimization results could further help standardize preparation of sequencing libraries from cfDNA samples.