title: High-Throughput Sequencing of Complete Mitochondrial Genomes.
abstract: Next-generation sequencing has revolutionized mitogenomics, turning a cottage industry into a high throughput process. This chapter outlines methodologies used to sequence, assemble, and annotate mitogenomes of non-model organisms using Illumina sequencing technology, utilizing either long-range PCR amplicons or gDNA as starting template. Instructions are given on how to extract DNA, conduct long-range PCR amplifications, generate short Sanger barcode tag sequences, prepare equimolar sample pools, construct and assess quality library preparations, assemble Illumina reads using either seeded reference mapping or de novo assembly, and annotate mitogenomes in the absence of an automated pipeline. Notes and recommendations, derived from our own experience, are given throughout this chapter.
authors.affiliation: Department of Life Sciences, Natural History Museum, Cromwell Road, London, SW7 5BD, UK. a.briscoe@nhm.ac.uk.
authors.forename: Andrew George
authors.initials: AG
authors.lastname: Briscoe
authors.affiliation: Department of Life Sciences, Natural History Museum, Cromwell Road, London, SW7 5BD, UK.
authors.forename: Kevin Peter
authors.initials: KP
authors.lastname: Hopkins
authors.affiliation: Department of Life Sciences, Natural History Museum, Cromwell Road, London, SW7 5BD, UK.
authors.forename: Andrea
authors.initials: A
authors.lastname: Waeschenbach
mesh_terms: D054629:Genome, Mitochondrial / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*
keywords: Illumina; Long-range PCR; MiSeq; Mitogenomics; Multiplexing; Shotgun sequencing