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Updated: Jun 9, 2026
Multiplexed Analysis of Retinal Gene Expression and Chromatin Accessibility Using scRNA-Seq and scATAC-Seq
Published on: March 12, 2021
Simultaneous mutation detection in 90 retinal disease genes in multiple patients using a custom-designed 300-kb
Judith C Booij1, Arne Bakker, Jamilia Kulumbetova
1Department of Clinical and Molecular Ophthalmogenetics, Netherlands Institute for Neuroscience, an institute of the Royal Netherlands Academy of Arts and Sciences, Amsterdam, The Netherlands.
Purpose:
To develop a high-throughput, cost-effective diagnostic strategy for the identification of known and new mutations in 90 retinal disease genes.
Design:
Evidence-based study.
Participants:
Sixty patients with a variety of retinal disorders, including Leber's congenital amaurosis, ocular albinism, pseudoxanthoma elasticum, retinitis pigmentosa, and Stargardt's disease.
Methods:
We designed a custom 300-kb resequencing chip. Polymerase chain reaction (PCR) amplification, DNA fragmentation, and chip hybridization were performed according to Affymetrix recommendations. Hybridization signals were analyzed using Sequence pilot module seq-C mutation detection software (2009). This resequencing approach was validated by Sanger sequence technology.
Main Outcome Measures:
Disease-causing sequence changes.
Results:
We developed a retinal resequencing chip that covers all exons of 90 retinal disease genes. We developed and tested multiplex primer sets for 1445 amplicons representing the genes included on the chip. We validated our approach by screening 87 exons from 25 retinal disease genes containing 87 known sequence changes previously identified in our patient group using Sanger sequencing. Call rates for successfully hybridized amplicons were 98% to 100%. Of the known single nucleotide changes, 99% could be detected on the chip. As expected, deletions could not be detected reliably.
Conclusions:
We designed a custom resequencing chip that can detect known and new sequence changes in 90 retinal disease genes using a new high-throughput strategy with a high sensitivity and specificity for one tenth of the cost of conventional direct sequencing. The developed amplification strategy allows for the pooling of multiple patients with non-overlapping phenotypes, enabling many patients to be analyzed simultaneously in a fast and cost-effective manner.
