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%. 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.

Figure 1.

Gene panel coverage. Boxplots illustrating the coverage obtained for all samples and the percent of bases covered at 100x across all six test centers. As illustrated, high coverage was obtained for the majority of samples across all centers, however a few samples had less than 90% of bases within the targeted regions of interest (ROI) obtaining >100x coverage. Notably, the partner center utilizing the same technology obtained high coverage for these particular samples across all ROI indicating that the low coverage arose from a local issue rather than an inherent technological or design issue. For the Multiplicom CLL MASTR kit, 24 samples were sequenced over two runs and this reduction in samples/run combined with the smaller size of the panel was reflected in the increase in median coverage obtained compared to the other assays whereby all 48 samples were sequenced in a single run. C1-C6 represent the six participating centers: C1 & C2 (Multiplicom), C3 & C4 (Illumina TSCA) and C5 & C6 (HaloPlex).

Figure 2.

Pairwise comparison of mutations detected by centers utilizing the Multiplicom, Illumina TSCA and HaloPlex assays. Mutation plots depicting the concordance and accuracy (narrow range) of the variant allele frequency (VAF) of variants found in this study. Variants are listed on the y-axis and variants detected by each center are denoted by different shapes. The VAF scale is on the x-axis and a VAF difference of less than 10% between partner centers is denoted by black shapes while red shapes indicate a VAF difference of greater than 10% between partner centers.

Figure 3.

TP53, SF3B1, and NOTCH1 variants detected by the six centers. Heatmaps and boxplots illustrating mutations within TP53, SF3B1, and NOTCH1. Columns represent the six test centers (C1-C6) and rows correspond to a mutation found in a specific sample. Color coding indicates the type of mutation whereas the shading depicts the variant allele frequency (VAF) (lighter to darker tones correspond to an increasing VAF). Boxes marked with a hash symbol (#) indicate that the variant was not detected by that particular center. TP53 splice site mutations resulted in the following nucleotide changes: c.96+1G>T, c.673-2A>T, c.783-2A>C and c.920-2A>G). C1 & C2 (Multiplicom), C3 & C4 (Illumina TSCA) and C5 & C6 (HaloPlex).