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Open Access 01-12-2017 | Research article

Applying next generation sequencing with microdroplet PCR to determine the disease-causing mutations in retinal dystrophies

Authors: Xinjing Wang, Wadih M. Zein, Leera D’Souza, Chimere Roberson, Keith Wetherby, Hong He, Angela Villarta, Amy Turriff, Kory R. Johnson, Yang C. Fann

Published in: BMC Ophthalmology | Issue 1/2017

Abstract

Background

Inherited Retinal dystrophy (IRD) is a broad group of inherited retinal disorders with heterogeneous genotypes and phenotypes. Next generation sequencing (NGS) methods have been broadly applied for analyzing patients with IRD. Here we report a novel approach to enrich the target gene panel by microdroplet PCR.

Methods

This assay involved a primer library which targeted 3071 amplicons from 2078 exons comprised of 184 genes involved in retinal function and/or retinal development. We amplified the target regions using the RainDance target enrichment PCR method and sequenced the products using the MiSeq NGS platform.

Results

In this study, we analyzed 82 samples from 67 families with IRD. Bioinformatics analysis indicated that this procedure was able to reach 99% coverage of target sequences with an average sequence depth of reads at 119×. The variants detected by this study were filtered, validated, and prioritized by pathogenicity analysis. Genotypes and phenotypes were correlated by determining a consistent relationship in 38 propands (56.7%). Pathogenic variants in genes related to retinal function were found in another 11 probands (16.4%), but the clinical correlations showed inconsistencies and insufficiencies in these patients.

Conclusions

The application of NGS in IRD clinical molecular diagnosis provides a powerful approach to exploring the etiology and pathology in patients. It is important for the clinical laboratory to interpret the molecular findings in the context of patient clinical presentations because accurate interpretation of pathogenic variants is critical for delivering solid clinical molecular diagnosis to clinicians and patients and improving the standard care of patients.

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Title: Applying next generation sequencing with microdroplet PCR to determine the disease-causing mutations in retinal dystrophies
Authors: Xinjing Wang
Wadih M. Zein
Leera D’Souza
Chimere Roberson
Keith Wetherby
Hong He
Angela Villarta
Amy Turriff
Kory R. Johnson
Yang C. Fann
Publication date: 01-12-2017
Publisher: BioMed Central
Published in: BMC Ophthalmology / Issue 1/2017
Electronic ISSN: 1471-2415
DOI: https://doi.org/10.1186/s12886-017-0549-5

Keynote webinar | Spotlight on sleep in brain health

Springer Medicine

Applying next generation sequencing with microdroplet PCR to determine the disease-causing mutations in retinal dystrophies

Abstract

Background

Methods

Results

Conclusions

Keynote webinar | Spotlight on sleep in brain health

Springer Medicine

Abstract

Background

Methods

Results

Conclusions

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