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

Jordi Maggi; Lisa Roberts; Samuel Koller; George Rebello; Wolfgang Berger; Rajkumar Ramesar

doi:10.3390/genes11070800

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 (Basel). 2020 Jul 15;11(7):800. doi: 10.3390/genes11070800.

Authors

Jordi Maggi^{1

2}, Lisa Roberts³, Samuel Koller¹, George Rebello³, Wolfgang Berger^{1

2

4}, Rajkumar Ramesar³

Affiliations

¹ Institute of Medical Molecular Genetic, University of Zurich, 8952 Schlieren, Switzerland.
² Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, 8006 Zurich, Switzerland.
³ University of Cape Town/MRC Genomic and Precision Medicine Research Unit, Division of Human Genetics, Department of Pathology, Institute of Infectious Disease and Molecular Medicine (IDM), Faculty of Health Sciences, University of Cape Town, Cape Town 7925, South Africa.
⁴ Neuroscience Center Zurich (ZNZ), University and ETH Zurich, 8006 Zurich, Switzerland.

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.

Keywords: ORF15; RP; RPGR; de novo assembly; diagnostics; genetic testing; secondary analysis.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Black People / genetics
Contig Mapping / methods
Contig Mapping / standards
Exons
Eye Proteins / genetics*
Female
Genetic Testing / methods*
Genetic Testing / standards
High-Throughput Nucleotide Sequencing / methods*
High-Throughput Nucleotide Sequencing / standards
Humans
Indigenous Peoples / genetics
Male
Mutation
Pedigree
Retinitis Pigmentosa / diagnosis
Retinitis Pigmentosa / genetics*
Sensitivity and Specificity
Sequence Analysis, DNA / methods
Sequence Analysis, DNA / standards

Substances

Eye Proteins
RPGR protein, human