High-Throughput Sequencing of Complete Mitochondrial Genomes

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Next-generation sequencing (NGS) has transformed mitogenomics into a high-throughput field.
• Established protocols for sequencing and assembling mitogenomes of non-model organisms are crucial for biodiversity and evolutionary studies.

Purpose of the Study:

• To provide a comprehensive methodological guide for sequencing, assembling, and annotating mitogenomes from non-model organisms.
• To detail the application of Illumina sequencing technology for mitogenome reconstruction.

Main Methods:

• DNA extraction from various sources.
• Long-range PCR amplification of mitochondrial DNA (mtDNA) or use of genomic DNA (gDNA).
• Illumina library preparation, quality assessment, and sequencing.
• Mitogenome assembly using seeded reference mapping or de novo approaches.
• Manual annotation of assembled mitogenomes.

Main Results:

• A detailed workflow for generating high-quality mitogenome data from non-model organisms.
• Successful assembly and annotation strategies applicable even without automated pipelines.
• Practical recommendations based on extensive laboratory experience.

Conclusions:

• The described methodologies enable efficient and accurate mitogenome sequencing and assembly for diverse non-model species.
• This chapter serves as a valuable resource for researchers entering the field of comparative mitogenomics.