In-Solution Hybridization for the Targeted Enrichment of the Whole Mitochondrial Genome.
A detailed protocol is presented for the targeted enrichment of whole mitochondrial genomes based on an in-solution hybridization strategy. Bait is produced in-house by sonication of two long-range PCR amplicons and ligation of biotinylated double-stranded adapters. Indexed target DNA is hybridized with the bait in a multiplex enrichment reaction and pulled down using magnetic streptavidin beads followed by subsequent post-enrichment PCR and sequencing on an Illumina MiSeq. This strategy removes the need for expensive commercial bait probes while allowing enrichment of multiple samples in a single hybridization reaction. The method is particularly suitable for degraded DNA as it is able to enrich short DNA fragments and is not susceptible to polymerase artifacts introduced during PCR-based assays.
Department of Imaging and Pathology, Forensic Biomedical Sciences, KU Leuven-University of Leuven, 3000, Leuven, Belgium. bram.bekaert@kuleuven.be.|Laboratory of Forensic Genetics and Molecular Archaeology, Department of Forensic Medicine, University Hospitals Leuven, KU Leuven-University of Leuven, Kapucijnenvoer 33 blok I, Box 7001, 3000, Leuven, Belgium. bram.bekaert@kuleuven.be.
B
B
Bekaert
Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK.
R
R
Ellerington
Faculty of Medicine and Pharmacy, Free University of Brussels, 1090, Brussels, Belgium.
L
L
Van den Abbeele
Department of Imaging and Pathology, Forensic Biomedical Sciences, KU Leuven-University of Leuven, 3000, Leuven, Belgium.|Laboratory of Forensic Genetics and Molecular Archaeology, Department of Forensic Medicine, University Hospitals Leuven, KU Leuven-University of Leuven, Kapucijnenvoer 33 blok I, Box 7001, 3000, Leuven, Belgium.
R
R
Decorte
D001710:Biotin / Q000737:chemistry; D053803:Forensic Genetics; D054629:Genome, Mitochondrial; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods
D001710:Biotin
Illumina MiSeq; In-solution hybridization; Mitochondrial DNA; Targeted enrichment