Table 1.
List of genes targeted in the PrismGuide IRD Panel System.
The target regions include all exons of 82 genes and an intron between exon 26 and 27 in CEP290, where pathogenic variants are observed [33].
Fig 1.
Workflow used with the PrismGuide IRD Panel System for genomic profiling.
(A) Genomic DNA was extracted from routine peripheral whole-blood samples. (B) Sequencing libraries were prepared using the PrismGuide IRD Panel Kit with ≥1000 ng of genomic DNA, and 82 genes related to IRDs were enriched by hybrid capture. The quality of each sequencing library was evaluated by DNA quantification. (C) DNA sequencing was performed using the Food and Drug Administration-regulated Illumina MiSeqDx System. (D) Germline variants for single-nucleotide variants (SNVs), small insertions and deletions (indels), and copy-number loss were called using the PrismGuide IRD Panel Analysis Program. The SNVs and small indels were annotated in terms of their pathogenicity according to the ACMG/AMP guidelines by the PrismGuide IRD Panel Analysis Program [31]. (E) Clinical reports were generated using the PrismGuide IRD Panel Analysis Program. The "Summary report” included the status of QC analysis and pathogenic and likely pathogenic SNV and small indels, classified using the five-tier system mentioned in the ACMG/AMP guidelines [31]. The “Sequencing report” included all variants detected and information pertaining to the analysis program and databases used.
Fig 2.
Accuracy of variant detection with PrismGuide IRD Panel System, compared to Sanger sequencing.
(A) The TP, FN, FP, TN, PPA, and NPA found for the variants of interest are indicated. SNV, single-nucleotide variant; indel, insertion and deletion; TP, true positive; FN, false negative; FP, false positive; TN, true negative; PPA, positive-percent agreement; NPA, negative-percent agreement; CI, confidence interval. (B) Number of variant reads with the indicated GC-content levels is shown in the boxplot. Each cross represents the mean of the reads. (C) The zygosity of the variants determined with the PrismGuide IRD Panel System was compared with that observed via Sanger sequencing. The numbers of homozygous/hemizygous and heterozygous variants and concordance rate are indicated in the two-by-two contingency table. Homo, homozygote; Hemi, hemizygote; hetero, heterozygote.
Table 2.
Accuracy of copy-number loss detection.
Fig 3.
Precision of variant detection with the PrismGuide IRD Panel System.
(A) The PPA values for detecting SNV and small indels with the PrismGuide IRD Panel System are shown with respect to repeatability and reproducibility. Percentages of agreement of 1,584 SNVs and 576 small indels in 144 replicates of NA24385 samples were indicated. SNV, single-nucleotide variant; indel, insertion and deletion; PPA, positive-percent agreement; n, Total number of SNVs or small indels tested in 144 replicates. (B) The graph shows the variant allele frequencies of the indicated variants. Error bars, standard deviation of 144 replicates.
Fig 4.
Accuracy of the detection of RPGR ORF15 variants with the PrismGuide IRD Panel System.
(A) Reads generated with the PrismGuide IRD Panel System for RPGR ORF15 (NM_001034853.2: c.1754–c.3459) in one patient, S736, are indicated by the gray peaks. The genomic organization of RPGR ORF15 is shown in the blue box. The dashed line indicates a 20× read depth. (B) Schematic representation of the transcript of a splice isoform of RPGR ORF15. Exons 1–14 and ORF15 are indicated by the light and dark gray boxes, respectively. The ORF15 variants from five patients with IRDs are also indicated. (C) Accuracy of variant detection with the PrismGuide IRD Panel System, compared with that of Sanger sequencing. The variants detected, variant allele frequency, read depth of the variant, and mean read depth in the ORF15 region (NM_001034853.2: c.1754–c.3459) are indicated for the PrismGuide IRD Panel System. The percentage of bases with a depth of ≥20× in ORF 15 is also shown. M, male; F, female; VAF, variant allele frequency.
Fig 5.
Effect of interfering substances on variant detection in the PrismGuide IRD Panel System.
(A) The percentages of variant detection in the presence of the indicated interfering substances were shown. The percentage of variant detection was determined by comparing the number of variants detected in the presence of the interfering substances in three clinical samples with that found in the control condition, where no interfering substances were added. The numbers of the variants detected are shown in parentheses. SNV, single-nucleotide variant; indel, insertion and deletion; EDTA, ethylenediaminetetraacetic acid. (B) Variant allele frequencies of the representative variants in the presence of the interfering substances are shown. The control condition involved sequencing in the absence of the interfering substances.
Fig 6.
Stability of peripheral whole blood and genomic DNA samples for variant detection with the PrismGuide IRD Panel System.
(A) The percentages of variant detection under the indicated storage conditions were shown. The percentage of variant detection was determined by comparing the number of variants detected in each storage condition in 11 clinical samples to that found in the control condition, where peripheral blood and DNA were immediately processed for library preparation without an extended storage period or unusual temperature conditions. Numbers of the variants are shown in parentheses. SNV, single-nucleotide variant; indel, insertion and deletion. (B) Variant allele frequencies of the representative variants observed in peripheral whole-blood and genomic-DNA samples after storage under the indicated conditions.