Background Inherited retinal diseases (IRDs) are a clinically and genetically heterogeneous set of disorders, for which diagnostic second-generation sequencing (next-generation sequencing, NGS) services have been developed worldwide. Methods We present the molecular findings of 537 individuals referred to a 105-gene diagnostic NGS test for IRDs. We assess the diagnostic yield, the spectrum of clinical referrals, the variant analysis burden and the genetic heterogeneity of IRD. We retrospectively analyse disease-causing variants, including an assessment of variant frequency in Exome Aggregation Consortium (ExAC). Results Individuals were referred from 10 clinically distinct classifications of IRD. Of the 4542 variants clinically analysed, we have reported 402 mutations as a cause or a potential cause of disease in 62 of the 105 genes surveyed. These variants account or likely account for the clinical diagnosis of IRD in 51% of the 537 referred individuals. 144 potentially disease-causing mutations were identified as novel at the time of clinical analysis, and we further demonstrate the segregation of known disease-causing variants among individuals with IRD. We show that clinically analysed variants indicated as rare in dbSNP and the Exome Variant Server remain rare in ExAC, and that genes discovered as a cause of IRD in the post-NGS era are rare causes of IRD in a population of clinically surveyed individuals. Conclusions Our findings illustrate the continued powerful utility of custom-gene panel diagnostic NGS tests for IRD in the clinic, but suggest clear future avenues for increasing diagnostic yields.

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BackgroundThe Azadirachta indica (neem) tree is a source of a wide number of natural products, including the potent biopesticide azadirachtin. In spite of its widespread applications in agriculture and medicine, the molecular aspects of the biosynthesis of neem terpenoids remain largely unexplored. The current report describes the draft genome and four transcriptomes of A. indica and attempts to contextualise the sequence information in terms of its molecular phylogeny, transcript expression and terpenoid biosynthesis pathways. A. indica is the first member of the family Meliaceae to be sequenced using next generation sequencing approach.ResultsThe genome and transcriptomes of A. indica were sequenced using multiple sequencing platforms and libraries. The A. indica genome is AT-rich, bears few repetitive DNA elements and comprises about 20,000 genes. The molecular phylogenetic analyses grouped A. indica together with Citrus sinensis from the Rutaceae family validating its conventional taxonomic classification. Comparative transcript expression analysis showed either exclusive or enhanced expression of known genes involved in neem terpenoid biosynthesis pathways compared to other sequenced angiosperms. Genome and transcriptome analyses in A. indica led to the identification of repeat elements, nucleotide composition and expression profiles of genes in various organs.ConclusionsThis study on A. indica genome and transcriptomes will provide a model for characterization of metabolic pathways involved in synthesis of bioactive compounds, comparative evolutionary studies among various Meliaceae family members and help annotate their genomes. A better understanding of molecular pathways involved in the azadirachtin synthesis in A. indica will pave ways for bulk production of environment friendly biopesticides.

Although a common cause of disease, copy number variants (CNVs) have not routinely been identified from next-generation sequencing (NGS) data in a clinical context. This study aimed to examine the sensitivity, and specificity of a widely used software package, ExomeDepth, to identify CNVs from targeted NGS datasets. We benchmarked the accuracy of CNV detection using ExomeDepth v1.1.16 applied to targeted NGS datasets, through comparison to CNV events detected through whole genome sequencing (WGS) for 25 individuals, and determined the sensitivity and specificity of ExomeDepth applied to these targeted NGS datasets to be 100% and 99.8%, respectively. To define quality assurance metrics for CNV surveillance through Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Corresponding Author: Graeme CM Black, graeme.black@manchester.ac.uk. Competing interestsThe authors of this manuscript have no competing interests to declare.Authors' contributions JME, GCMB, SCR and BP designed and coordinated the study. JME, CC, StB, SaB, SG, RLG, PIS, BH, MM, ARW, WGN, SCR, GCMB contributed genetic and/or phenotypic data. JME wrote the manuscript and all authors provided important revisions and intellectual content. ExomeDepth, we undertook simulation of single exon (n=1000) and multiple-exon heterozygous deletion events (n=1749), determining a sensitivity of 97% (n=2749). We identified that the extent of sequencing coverage, the inter-and intra-sample variability in the depth of sequencing coverage, and the composition of analysis regions are all important determinants of successful CNV surveillance through ExomeDepth. We then applied these quality assurance metrics during CNV surveillance for 140 individuals across 12 distinct clinical areas, encompassing over 500 potential rare disease diagnoses. All 140 individuals lacked molecular diagnoses after routine clinical NGS testing, and through application of ExomeDepth we identified 17 CNVs contributing to the cause of a Mendelian disorder. Our findings support the integration of CNV detection using ExomeDepth v1.1.16 with routine targeted NGS diagnostic services for Mendelian disorders. Implementation of this strategy increases diagnostic yields and enhances clinical care. Europe PMC Funders Group