Discovering causative genetic variants in individual cases of suspected 
mitochondrial disease requires interrogation of both the mitochondrial (mtDNA) 
and nuclear genomes. Whole-exome sequencing can support simultaneous dual-genome 
analysis, although currently available capture kits do not target the mtDNA 
genome and provide insufficient capture for some nuclear-encoded mitochondrial 
genes. To optimize interrogation of nuclear and mtDNA genes relevant to 
mitochondrial biology and disease, a custom SureSelect "Mito-Plus" whole-exome 
library was formulated by blending RNA "baits" from three separate designs: (A) 
Agilent Technologies SureSelectXT 50 Mb All Exon PLUS Targeted Enrichment Kit, 
(B) 16-gene nuclear panel targeting sequences for known MitoCarta proteins not 
included in the 50 Mb All Exon design, and (C) sequences targeting the entire 
mtDNA genome. The final custom formulations consisted of a 1:1 ratio of nuclear 
baits to which a 1 to 1,000-fold diluted ratio of mtDNA genome baits were 
blended. Patient sample capture libraries were paired-end sequenced on an 
Illumina HiSeq 2000 system using v3.0 SBS chemistry. mtDNA genome coverage 
varied depending on the mtDNA:nuclear blend ratio, where a 1:100 ratio provided 
optimal dual-genome coverage with 10X coverage for over 97.5% of all targeted 
nuclear regions and 1,000X coverage for 99.8% of the mtDNA genome. mtDNA 
mutations were reliably detected to at least an 8% heteroplasmy level, as 
discriminated both from sequencing errors and potential contamination from 
nuclear mtDNA transcripts (Numts). The "1:100 Mito-Plus Whole-Exome" Agilent 
capture kit offers an optimized tool for whole-exome analysis of nuclear and 
mtDNA genes relevant to the diagnostic evaluation of mitochondrial disease.