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

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%. 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 VAF >0.5%. Reproducibility was assessed by looking at the inter-laboratory variation in detecting mutations and 107/115 (93% concordance) of mutations were detected by all 6 centers, while the remaining 8/115 (7%) variants were undetected by a single center and 6/8 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 VAFs >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.

journal_name

Haematologica

journal_title

Haematologica

authors

Sutton LA,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,Ca

doi

10.3324/haematol.2019.234716

subject

Has Abstract

pub_date

2020-04-09 00:00:00

eissn

0390-6078

issn

1592-8721

pii

haematol.2019.234716

pub_type

杂志文章

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