Comparative analysis of targeted next-generation sequencing panels for the detection of gene mutations in chronic lymphocytic leukemia: an ERIC multi-center study.
Haematologica(2021)
Abstract
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.
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