Long-Read Whole-Genome Sequencing as a Tool for Variant Detection in Inherited Retinal Dystrophies

Abstract

Advances in whole-genome sequencing (WGS) have significantly enhanced our ability to detect genomic variants underlying inherited diseases. In this study, we performed long-read WGS on 24 patients with inherited retinal dystrophies (IRDs) to validate the utility of nanopore sequencing in detecting genomic variations. We confirmed the presence of all previously detected variants and demonstrated that this approach allows for the precise refinement of structural variants (SVs). Furthermore, we could perform genotype phasing by sequencing only the probands, confirming that the variants were inherited in trans. Moreover, nanopore sequencing enables the detection of complex variants, such as transposon insertions and structural rearrangements. This comprehensive assessment illustrates the power of long-read sequencing in capturing diverse forms of genomic variation and in improving diagnostic accuracy in IRDs.

Keywords: inherited retinal dystrophies; long-read sequencing; nanopore sequencing.

Figure 1

Distribution of previously reported variants by type (A) and mutation consequence (B). (C) IGV visualization of a previously reported indel in the C001VXB patient, genomic location is surrounded by a red rectangle. (D) Coverage distribution of LR-WGS data in the chr16:10000000–25000000 region containing the previously detected duplication of the C00231A patient (red) compared to the C00231G patient (gray), which was considered a negative control. Possible duplication boundaries are marked with a dotted line.

Figure 2

(A) Pedigree of C001VXB patient and his parents. Squares indicate males, circles indicate females, and the arrow denotes the proband. (B) Visualization of detected deletion in the EYS gene using MLPA analysis. (C) Ribbon visualization of the deletion in LR-WGS data. In the top image, the red rectangle frames both breakpoints with split reads on both sides. The bottom image shows the alignment of a selected single read that contains the deletion.

Figure 3

(A) Pedigree of C002312 patient and his parents. Squares indicate males, circles indicate females, and the arrow denotes the proband. Sanger segregation results are shown underneath each individual. (B) Ribbon visualization of the deletion. In the top image, the red rectangle frames both breakpoints, showing split reads on both sides. The bottom image displays the alignment of a selected single read that contains the deletion. (C) Visualization of sequences aligned with CFAP20 on IGV. Reads containing the SNV, framed on a red rectangle, are highlighted in light blue. Reads containing the deletion breakpoint, indicated by a red line, are highlighted in light orange.

Figure 4

(A) Pedigree of C00231G patient and his parents. Squares indicate males, circles indicate females, and the arrow denotes the proband. Sanger segregation results are shown underneath each individual. (B) Ribbon visualization of the deletion. In the top image, the red rectangle frames both breakpoints, showing split reads on both sides. The bottom image displays the alignment of a selected single read that contains the deletion. (C) Visualization of sequences aligned with MYO7A on IGV. Reads are grouped and colored by haplotype. On the right image, the red rectangle frames both breakpoints of the deletion, showing split reads on both sides.

Figure 5

(A) Pedigree of the C001VWE patient. Squares indicate males, circles indicate females, and the arrow denotes the proband. (B) Ribbon visualization of a selected read containing the inverted insertion. (C) Schematic representation of chromosome 1 reorganization detected in the patient.

Figure 6

Schematic representation of the recombinant X chromosome rearrangement identified in the C001VWE patient. The region sizes are described above. Colored breakpoints were called, and black breakpoints were identified upon manual exploration.