In-Solution Hybridization for the Targeted Enrichment of the Whole Mitochondrial Genome

B Bekaert; R Ellerington; L Van den Abbeele; R Decorte

doi:10.1007/978-1-4939-3597-0_14

In-Solution Hybridization for the Targeted Enrichment of the Whole Mitochondrial Genome

Methods Mol Biol. 2016:1420:173-83. doi: 10.1007/978-1-4939-3597-0_14.

Authors

B Bekaert^{1

2}, R Ellerington³, L Van den Abbeele⁴, R Decorte^{5

6}

Affiliations

¹ Department of Imaging and Pathology, Forensic Biomedical Sciences, KU Leuven-University of Leuven, 3000, Leuven, Belgium. bram.bekaert@kuleuven.be.
² 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.
³ Faculty of Health and Medical Sciences, University of Surrey, Guildford, GU2 7XH, UK.
⁴ Faculty of Medicine and Pharmacy, Free University of Brussels, 1090, Brussels, Belgium.
⁵ Department of Imaging and Pathology, Forensic Biomedical Sciences, KU Leuven-University of Leuven, 3000, Leuven, Belgium.
⁶ 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.

PMID: 27259740
DOI: 10.1007/978-1-4939-3597-0_14

Abstract

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.

Keywords: Illumina MiSeq; In-solution hybridization; Mitochondrial DNA; Targeted enrichment.

MeSH terms

Biotin / chemistry
Forensic Genetics
Genome, Mitochondrial*
Humans
Nucleic Acid Hybridization / methods*
Polymerase Chain Reaction / methods

Substances

Biotin