Fol. Biol. 2025, 71, 244-255
Development and Technical Validation of the Clinical Lymphoma Exploration and Research Sequencing (CLEARS) Panel and Its Optimization for ctDNA Analysis
, Adriana Veľasová1, Petra Zemánková3, Petr Nehasil3,4,5, Kristýna Kupcová1, Jana Soukupová3, Markéta Janatová3, Milena Hovhannisyan3, Jana Seňavová1,2, Anton Tkachenko1, Marek Trněný2, Ondřej Havránek1,2
1BIOCEV, First Faculty of Medicine, Charles University, Vestec, Czech Republic
2First Department of Medicine – Department of Haematology, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
3Institute of Medical Biochemistry and Laboratory Diagnostics, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
4Department of Paediatrics and Inherited Metabolic Disorders, First Faculty of Medicine, Charles University and General University Hospital in Prague, Prague, Czech Republic
5Institute of Pathological Physiology, First Faculty of Medicine, Charles University, Prague, Czech Republic
Received July 4, 2025
Accepted December 1, 2025
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.
Supplementary materials - Figures and Table 1: Figures (pdf), Table 1 (xlsx)
Keywords
liquid biopsy, circulating tumour DNA, ctDNA, CAPP-Seq, next-generation DNA sequencing, lymphoma, non-Hodgkin lymphoma, NHL, B-NHL, T-NHL, Hodgkin lymphoma, HL, CLEARS.
Funding
This study was supported by the Ministry of Health of the Czech Republic (Czech Health Research Council grants – NU21-03-00411, NU22-03-00182, DRO – VFN00064165), the National Institute for Cancer Research (EXCELES – LX22NPO5102), Charles University (SVV 260637), and by institutional research project BBMRI_CZ LM2023033.
References
Copyright
This is an open-access article distributed under the terms of the Creative Commons Attribution License.