MitoExome: A Custom Whole Exome Kit for Mitochondrial Disease Research and Diagnosis that Captures All MitoCarta Genes and the Mitochondrial Genome. X. Gai1, E. A. Pierce2, M. Consugar2, M. Lvova3, D. C. Wallace3, E. LeProust4, M. J. Falk5 1) Center for Biomedical Informatics, Loyola University Stritch School of Medicine, Maywood, IL; 2) Ocular Genomics Institute and Berman-Gund Laboratory for the Study of Retinal Degenerations, Department of Ophthalmology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA; 3) Center for Mitochondrial and Epigenomic Medicine, Department of Pathology, The Childrens Hospital of Philadelphia, and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA; 4) Agilent Research and Development, CA; 5) Divisions of Human Genetics, and Rehabilitation and Metabolic Disease, Department of Pediatrics, The Childrens Hospital of Philadelphia, and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA.

   Discovering mitochondrial disease variants and genes requires simultaneous interrogation of both the mitochondrial genome and the nuclear genome, which encodes at least 1100 mitochondria-targeted proteins whose exact composition varies between tissues. Currently available whole exome capture kits do not capture the mitochondrial genome and provide insufficient capture for some MitoCarta-catalogued nuclear mitochondrial genes. METHODS: To improve upon this, we formulated a custom SureSelect library by blending RNA baits from three separate designs: (A) Agilent Technologies (Santa Clara, CA) SureSelectXT 50 Mb All Exon PLUS Targeted Enrichment Kit, (B) a panel of 16 nuclear-encoded mitochondrial genes that target sequences not included in the All-Exon design , and (C) sequences targeting the entire mitochondrial DNA genome. Designs B and C were created in eArray by standard 1X tiling across the target hg19 mitochondrial loci or 2X tiling across nuclear genes. The final custom formulations consisted of a 1:1 ratio of Design A/All-Exon to Design B baits. Design C/mitochondrial baits were subsequently blended in either at equal molar ratio or reduced concentrations by 10, 50, 100, 200, 500, or 1000 fold less than the nuclear baits. Patient sample capture libraries were 2 x 101 bp paired-end sequenced on an Illumina (SanDiego, CA) HiSeq 2000 Next-Generation Sequencing system using v3.0 SBS chemistry. RESULTS: As expected, the mitochondrial genome coverage varied depending on the mitochondrial:nuclear blend ratio, with 1:100 and 1:200 ratios providing the optimal coverage for both genomes. 1X, 10X, or 20X coverage was achieved for over 99%, 97%, and 95% of all targeted nuclear exons. 1X, 10X, 100X, and 1000X coverage was obtained on average for 100%, 99.99%, 99.9%, and 99% of the mitochondrial genome. Low-level heteroplasmy was sensitively detected. Analysis of nuclear-encoded mitochondrial transcripts is ongoing. CONCLUSIONS: This custom mito-plus whole exome capture kit allows simultaneous detection of both nuclear and mitochondrial DNA genomes, including sensitive detection of low-level heteroplasmy mtDNA variants that commonly cause mitochondrial disease. It represents an ideal experimental platform for both research and clinical-based diagnostic investigations of suspected mitochondrial diseases.

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