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Ricardo Frausto, Jonathan Han, Anthony Aldave; Identification of Genetic Variant Candidates for Posterior Polymorphous Corneal Dystrophy 1 utilizing Next-Generation Sequencing. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4731.
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To identify the genetic basis of posterior polymorphous corneal dystrophy 1 (PPCD1) by whole-region capture and subsequent next-generation sequencing (NGS) of the 14.5Mbp PPCD1 locus on 20p12.1-q11.21.
Slit-lamp examination was performed on 29 members of a multigenerational PPCD pedigree. Peripheral blood samples were collected from each family member, and extracted genomic DNA samples from selected affected and unaffected individuals were used for NGS of the captured PPCD1 locus. Alignment of the paired-end reads was performed using the Bowtie2 algorithm, while single nucleotide variant (SNV) detection was performed using SAMtools mpileup, within the Partek Flow software. A list of variants identified within coding regions of the PPCD1 locus (bounded by the D20S182 and D20S195 genetic markers) was generated using the Partek Genomic Suite software. To determine the sensitivity and specificity of NGS at two different coverage levels in identifying SNVs in the PPCD1 locus, the identified variants in two unaffected individual were compared to those previously identified in the PPCD1 locus with Sanger sequencing.
Eleven individuals were classified as affected, while 18 individuals had an unremarkable ocular examination and were classified as unaffected. NGS of the captured PPCD1 locus was performed on genomic DNA from 4 affected and 4 unaffected individuals from this pedigree. Analysis of the sequencing data revealed 39 coding region SNVs present only in the affected samples. Twenty-six of the 39 SNVs were predicted to produce synonymous amino acid substitutions, while the remaining 13 were predicted to produce missense substitutions. Comparing variants that were previously identified using Sanger sequencing to NGS results demonstrated a sensitivity of 93.6% for NGS (132/141) at ≥5x coverage and 92.9% (131/141) at ≥10x coverage. The specificity of NGS for identification of SNVs was found to be 100% at both ≥5x or ≥10x coverage.
NGS provides a cost-effective, rapid means of resequencing a candidate gene interval to identify coding region variants. In comparison to Sanger sequencing, NGS provides high sensitivity and specificity, with the advantage of high-throughput analysis.
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