Next-generation sequencing (NGS) has transitioned from research to clinical routine, yet the comparability of different technologies for mutation profiling remains an open question. We performed a European multicenter (n=6) evaluation of three amplicon-based NGS assays targeting 11 genes recurrently mutated in chronic lymphocytic leukemia. Each assay was assessed by two centers using 48 pre-characterized chronic lymphocytic leukemia samples; libraries were sequenced on the Illumina MiSeq instrument and bioinformatics analyses were centralized. Across all centers the median percentage of target reads ≥100x ranged from 94.2- 99.8%. In order to rule out assay-specific technical variability, we first assessed variant calling at the individual assay level i.e., pairwise analysis of variants detected amongst partner centers. After filtering for variants present in the paired normal sample and removal of PCR/sequencing artefacts, the panels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex) concordance at a variant allele frequency (VAF) >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107 of 115 (93% concordance) mutations were detected by all six centers, while the remaining eight variants (7%) were undetected by a single center. Notably, 6 of 8 of these variants concerned minor subclonal mutations (VAF <5%). We sought to investigate low-frequency mutations further by using a high-sensitivity assay containing unique molecular identifiers, which confirmed the presence of several minor subclonal mutations. Thus, while amplicon-based approaches can be adopted for somatic mutation detection with VAF >5%, after rigorous validation, the use of unique molecular identifiers may be necessary to reach a higher sensitivity and ensure consistent and accurate detection of low-frequency variants.

AP HP Hopital Pitie Salpetriere Department of Hematology Sorbonne Université Paris Franc

Cancer Sciences Faculty of Medicine University of Southampton Southampton UK

Center of Molecular Medicine CEITEC Central European Institute of Technology Masaryk University Brno Czech Republi

Centro de Investigación Biomédica en Red de Cáncer Madrid Spain and Hospital Clínic of Barcelona Universitat de Barcelona Barcelona Spai

Clinical Genetics Karolinska University Laboratory Karolinska University Hospital Stockholm Swede

Department of Hematology Royal Bournemouth Hospital Bournemouth U

Department of Immunology Genetics and Pathology Science for Life Laboratory Uppsala University Uppsala Swede

Department of Internal Medicine 3 Ulm University Ulm German

Department of Molecular Medicine and Surgery Karolinska Institutet Stockholm Swede

Division of Experimental Oncology Università Vita Salute San Raffaele and IRCCS San Raffaele Scientific Institute Milan Ital

Hematology Department Oncology Institute of Southern Switzerland and Institute of Oncology Research Bellinzona Switzerlan

Institut d'Investigacions Biomèdiques August Pi iSunyer Barcelona Spain

Institute of Applied Biosciences Center for Research and Technology Thessaloniki Gree