Panel-based genetic diagnostic testing for inherited eye diseases is highly accurate and reproducible, and more sensitive for variant detection, than exome sequencing

Abstract

Purpose: Next-generation sequencing-based methods are being adopted broadly for genetic diagnostic testing, but the performance characteristics of these techniques with regard to test accuracy and reproducibility have not been fully defined.

Methods: We developed a targeted enrichment and next-generation sequencing approach for genetic diagnostic testing of patients with inherited eye disorders, including inherited retinal degenerations, optic atrophy, and glaucoma. In preparation for providing this genetic eye disease (GEDi) test on a CLIA-certified basis, we performed experiments to measure the sensitivity, specificity, and reproducibility, as well as the clinical sensitivity, of the test.

Results: The GEDi test is highly reproducible and accurate, with sensitivity and specificity of 97.9 and 100%, respectively, for single-nucleotide variant detection. The sensitivity for variant detection was notably better than the 88.3% achieved by whole-exome sequencing using the same metrics, because of better coverage of targeted genes in the GEDi test as compared with a commercially available exome capture set. Prospective testing of 192 patients with inherited retinal degenerations indicated that the clinical sensitivity of the GEDi test is high, with a diagnostic rate of 51%.

Conclusion: Based on quantified performance metrics, the data suggest that selective targeted enrichment is preferable to whole-exome sequencing for genetic diagnostic testing.

Figure 1

(A) Analysis of empiric GEDi data shows that design gaps ≤ 75 bp were relatively well covered by “near-target” capture. (B) Representative Depth-of-Coverage (DoC) plot for a 12x-multiplexed capture sample using the GEDi targeted enrichment kit and 2 × 121 bp paired-end sequenced using an Illumina MiSeq.

Figure 2

(A) Integrative Genomics Viewer (IGV) screenshot of representative GEDi NGS validation data at chr15:78397352. The Omni 2.5 SNP data were determined to be incorrect in all samples due to a single base deletion adjacent to the interrogated base that shifted the analyzed base. (B) IGV screenshot of the putative c.1028T>G mutation of FSCN2 in OGI-267-573, clarifying the false positive variant call was due to mis-alignment of some NGS sequencing reads. (C) GEDi vs. Omni 2.5 concordance histogram plot corresponding to the 2,443 shared SNPs between the GEDi design and Omni 2.5 SNP for all 36 replicates of the 4 validation samples used in this study. KEY: MATCH – All GEDi NGS replicates matched Omni 2.5 SNP data; NO CALL – no NGS result; NO MATCH – ≥ 1 NGS replicate did not match Omni 2.5 SNP data; OMNI NO VALUE – no Omni 2.5 SNP result; NO CALL/MATCH – ≥ 1 NGS replicate had no result; all other NGS replicates matched Omni 2.5 SNP data.

Figure 3

Comparison of V4+UTR WES and GEDi capture baits at the 5′-end of ABCC6. The ABCC6 reference used is a “collapsed” reference that accounts for all known gene isoforms.