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.