Mitochondrial disease genetic diagnostics: optimized whole-exome analysis for all MitoCarta nuclear genes and the mitochondrial genome Journal Article

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Authors: Falk, M. J.; Pierce, E. A.; Consugar, M.; Xie, M. H.; Guadalupe, M.; Hardy, O.; Rappaport, E. F.; Wallace, D. C.; LeProust, E.; Gai, X.
Article Title: Mitochondrial disease genetic diagnostics: optimized whole-exome analysis for all MitoCarta nuclear genes and the mitochondrial genome
Abstract: 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.
Journal Title: Discovery medicine
Volume: 14
Issue: 79
ISSN: 1944-7930; 1539-6509
Publisher: Unknown  
Journal Place: United States
Date Published: 2012
Start Page: 389
End Page: 399
Language: eng
Notes: GR: R03 DK082521/DK/NIDDK NIH HHS/United States; GR: R03-DK082446/DK/NIDDK NIH HHS/United States; JID: 101250006; ppublish