De Novo Assembly-Based Analysis of RPGR Exon ORF15 in an Indigenous African Cohort Overcomes Limitations of a Standard Next-Generation Sequencing (NGS) Data Analysis Pipeline.

Genes
Jordi MaggiR Ramesar

Abstract

RPGR exon ORF15 variants are one of the most frequent causes for inherited retinal disorders (IRDs), in particular retinitis pigmentosa. The low sequence complexity of this mutation hotspot makes it prone to indels and challenging for sequence data analysis. Whole-exome sequencing generally fails to provide adequate coverage in this region. Therefore, complementary methods are needed to avoid false positives as well as negative results. In this study, next-generation sequencing (NGS) was used to sequence long-range PCR amplicons for an IRD cohort of African ancestry. By developing a novel secondary analysis pipeline based on de novo assembly, we were able to avoid the miscalling of variants generated by standard NGS analysis tools. We identified pathogenic variants in 11 patients (13% of the cohort), two of which have not been reported previously. We provide a novel and alternative end-to-end secondary analysis pipeline for targeted NGS of ORF15 that is less prone to false positive and negative variant calls.

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Citations

Feb 7, 2021·International Journal of Molecular Sciences·Jordi MaggiWolfgang Berger
Jun 3, 2021·International Journal of Molecular Sciences·Adrian DockeryG Jane Farrar

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Methods Mentioned

BETA
exome sequencing
electrophoresis
PCR

Clinical Trials Mentioned

NCT03116113

Software Mentioned

MAFFT
GATK
GATK Genome Analysis ToolKit
Mutalyzer
Python script
gnomAD
Python
MEM
Bioedit
house

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