The Development of Next-Generation Sequencing Assays for the Mitochondrial Genome and 108 Nuclear Genes Associated with Mitochondrial Disorders

Abstract

Sanger sequencing of multigenic disorders can be technically challenging, time consuming, and prohibitively expensive. High-throughput next-generation sequencing (NGS) can provide a cost-effective method for sequencing targeted genes associated with multigenic disorders. We have developed a NGS clinical targeted gene assay for the mitochondrial genome and for 108 selected nuclear genes associated with mitochondrial disorders. Mitochondrial disorders have a reported incidence of 1 in 5000 live births, encompass a broad range of phenotypes, and are attributed to mutations in the mitochondrial and nuclear genomes. Approximately 20% of mitochondrial disorders result from mutations in mtDNA, with the remaining 80% found in nuclear genes that affect mtDNA levels or mitochondrion protein assembly. In our NGS approach, the 16,569-bp mtDNA is enriched by long-range PCR and the 108 nuclear genes (which represent 1301 amplicons and 680 kb) are enriched by RainDance emulsion PCR. Sequencing is performed on Illumina HiSeq 2000 or MiSeq platforms, and bioinformatics analysis is performed using commercial and in-house developed bioinformatics pipelines. A total of 16 validation and 13 clinical samples were examined. All previously reported variants associated with mitochondrial disorders were found in validation samples, and 5 of the 13 clinical samples were found to have mutations associated with mitochondrial disorders in either the mitochondrial genome or the 108 nuclear genes. All variants were confirmed by Sanger sequencing.

Current practices and guidelines for clinical next-generation sequencing oncology testing

“…The most prevalent current implementation of NGS for oncology is mutation detection via targeted panels [1][2][3][4][5] . These assays use molecular methods such as multiplex polymerase chain reactions (PCR) to isolate clinically relevant segments of the genome, such as mutation hotspots or coding exons of entire genes.…”

Targeted Next-Generation Sequencing: the Clinician’s Stethoscope for Genetic Disorders

“…At present, the targeted DNA sequencing platform from RainDance Technologies offers the possibility to successfully use up to 20,000 primer pairs in one experiment [17]. Excellent results using microdroplet-PCR followed by NGS have been achieved for the diagnostics of several heterogeneous disorders, such as congenital muscular dystrophies [53], hearing loss [54] or mitochondrial disorders [55]. Furthermore, compartmentalization of a highly multiplexed PCR can also be achieved by performing several thousands of individual reactions in separate chambers inside a microfluidic chip [48] (e.g., Access Array System from Fluidigm [47]).…”

Molecular diagnosis of coenzyme Q10deficiency

“…Regarding custom-targeted NGS, Dames et al assessed the diagnostic yield for mitochondrial disorders of a panel consisting of 108 nuclear genes in combination with mitochondrial DNA (mtDNA) sequencing, in order to cover both genomes [43]. Because mitochondrial disorders encompass a wide range of phenotypes and a large number of genes, NGS is an ideal method for variant detection.…”