Sutton L, Ljungström V, Enjuanes A, Cortese D, Skaftason A, Tausch E, Stano Kozubik K, Nadeu F, Armand M, Malcikova J, Pandzic T, Forster J, Davis Z, Oscier D, Rossi D, Ghia P, Strefford JC, Pospisilova S, Stilgenbauer S, Davi F, Campo E, Stamatopoulos K, Rosenquist R
Haematologica 106 (3) 682-691 [2021-03-01; online 2021-03-01]
Next-generation sequencing (NGS) has transitioned from research toclinical routine, yet the comparability of different technologies formutation profiling remains an open question. We performed aEuropean multicenter (n=6) evaluation of three amplicon-based NGS assaystargeting 11 genes recurrently mutated in chronic lymphocytic leukemia.Each assay was assessed by two centers using 48 pre-characterized chroniclymphocytic leukemia samples; libraries were sequenced on the IlluminaMiSeq instrument and bioinformatics analyses were centralized. Across allcenters the median percentage of target reads ≥100x ranged from 94.2-99.8%. In order to rule out assay-specific technical variability, we firstassessed variant calling at the individual assay level i.e., pairwise analysis ofvariants detected amongst partner centers. After filtering for variants presentin the paired normal sample and removal of PCR/sequencing artefacts, thepanels achieved 96.2% (Multiplicom), 97.7% (TruSeq) and 90% (HaloPlex)concordance at a variant allele frequency (VAF) >0.5%. Reproducibility wasassessed by looking at the inter-laboratory variation in detecting mutationsand 107 of 115 (93% concordance) mutations were detected by all six centers,while the remaining eight variants (7%) were undetected by a singlecenter. Notably, 6 of 8 of these variants concerned minor subclonal mutations(VAF <5%). We sought to investigate low-frequency mutations furtherby 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 mutationdetection with VAF >5%, after rigorous validation, the use of unique molecularidentifiers may be necessary to reach a higher sensitivity and ensureconsistent and accurate detection of low-frequency variants.
Clinical Genomics Uppsala [Service]
NGI Uppsala (SNP&SEQ Technology Platform) [Service]
National Genomics Infrastructure [Service]