Comparison of exon-level copy number variants in CytoScan XON assay and next-generation sequencing in clinical samples

•A CytoScan XON assay was developed to assess exon-level CNVs.•The assay detected 15 out of 23 small exon-level CNVs identified by NGS.•Three of the undetected exon level CNVs were false positives in the NGS results.•The assay could not detect the three exon-level CNVs in PKD1 and TSC2.•The assay is a promising complementary tool for the detection of exon-level CNVs. Next-generation sequencing (NGS)-based copy number variants (CNVs) have high false-positive rates. The fewer the exons involved, the higher the false-positive rate. A CytoScan XON assay was developed to assess exon-level CNVs. Twenty-three clinically relevant exon-level CNVs in 20 patient blood samples found in previous NGS studies were compared with the results from the CytoScan XON and multiplex ligation-dependent probe amplification (MLPA). Fifteen of the 23 exon-level CNVs were consistent with the NGS results. Among these, eight were confirmed using MLPA. In six out of eight discrepancies between the CytoScan Xon and NGS, MLPA was performed, and three were negative, indicating that the CNVs in NGS were false positives. The CytoScan XON exhibits a sensitivity of 72.7% for small exon-level CNVs, along with a specificity of 100%. The assay could not detect the three exon-level CNVs in PKD1 and TSC2 that were detected using both NGS and MLPA. This could be due to the distribution of the probes in some areas, and the CNV-calling regions containing multiple exons. The CytoScan XON assay is a promising complementary tool for the detection of exon-level CNVs, provided that the users carefully examine the distribution of probes and calling regions.