Comparison of four next generation sequencing platforms for fusion detection: Oncomine by ThermoFisher, AmpliSeq by illumina, FusionPlex by ArcherDX, and QIAseq by QIAGEN

Abstract

As fusion detection NGS techniques are adopted by clinical labs, assay performance comparison is urgently needed. We compared four fusion-detection assay platforms on a pilot cohort of 24 prostate cancer samples: (1) Oncomine Comprehensive panel v3; (2) AmpliSeq comprehensive panel v3; (3) The solid tumor panel of FusionPlex; and (4) The human oncology panel of QIAseq. The assays were compared for the detection of different types of fusion based on whether the partner gene or the breakpoints are known. All assays detected fusion with known gene partners and known breakpoint, represented by TMPRSS2-ERG. A fusion with known partners but unknown breakpoint, TMPRSS2-ETV4, was reported by OCAv3 and FusionPlex, but not by AICv3 because the specific breakpoint was not in the manifest, nor by QIAseq since the panel did not target the exact exons involved. For fusion with unknown partners, FusionPlex identified the largest number of ETV1 fusions because it had the highest exon coverage for ETV1. Among these, SNRPN-ETV1 and MALAT1-ETV1, were novel findings. To determine reportability of low-level calls of highly prevalent fusions, such as TMPRSS2-ERG, we propose the use of percent fusion reads over total number of reads per sample instead of the fusion read count.

Keywords: Assay comparison; ETS genes; Fusion; NGS; Prostate cancer.

Figure 1:

Fusions detected in the study and samples tested on each assay. Green cells in the top panel denote the presence of fusion. Grey dots in the lower panel indicate samples tested by each assay.

Figure 2:

The TMPRSS2-ETV4 fusion reported by OCAv3 and FusionPlex and verified using RT-PCR and sanger sequencing. (A) displays the fusion in Ion Reporter Genomic Viewer (IRGV) for sample 6. The marked horizontal line and the two segments on top denote the location of the fusion breakpoint known to the manifest. The nucleotide sequence used as the reference in the manifest is displayed at the bottom. The middle portion displays the alignment of all the fusion reads (light grey) in relation to the reference and examples of a few reads in light blue. (B) displays JBrowser view of the fusion breakpoint with the highest number of unique reads in sample 6: The “BED GSP2” track shows the position and direction of the target gene specific primers (GSP); The “BED Contigs” shows the contiguous consensus sequence; and the “BAM-SNPs/Coverage” track displays read coverage, which is followed by examples of individual sequencing reads that encompassed the fusion breakpoint; (C) displays sanger sequencing results of the RT-PCR amplicon targeting TMPRSS2-ETV4 and the fusion junction. All genomic coordinates are based on human genome build GRCh37/Hg19.

Figure 3:

The TMPRSS2-ERG fusion reported based on false positive cutoffs established using RT-PCR as a reference method. (A) OCAv3: percent of fusion read count over total mapped reads (%TMR); (B) AICv3: percent of fusion read count over total on-target aligned reads (%TAR); (C) FusionPlex: percent of unique fusion read count over total unique RNA reads; and (D) QIAseq: percent of unique fusion read count over total unique reads. The grey and black boxes under the X-axis denote the RT-PCR call for TMPRSS2-ERG fusion. Each black dot represents a sample. On each panel, the first group from the left with eight samples were used to define false-positive cutoffs, which were calculated as the average plus three times’ standard deviation. The cutoff value is noted as the grey dashed line. The second group (negative) and third group (positive) from the left demonstrated the validation of the cutoff on the remaining samples.