Recent methodological advances have transformed the field of ancient DNA (aDNA). Basic bioinformatics skills are becoming essential requirements to process and analyze the sheer amounts of data generated by current aDNA studies and in biomedical research in general. This chapter is intended as a practical guide to the assembly of ancient mitochondrial genomes, directly from genomic DNA-derived next-generation sequencing (NGS) data, specifically in the absence of closely related reference genomes. In a hands-on tutorial suitable for readers with little to no prior bioinformatics experience, we reconstruct the mitochondrial genome of a woolly mammoth deposited ~45,000 years ago. We introduce key software tools and outline general strategies for mitogenome assembly, including the critical quality assessment of assembly results without a reference genome.
Keywords: Ancient DNA; Bioinformatics; Genome assembly; Mitogenomics; Next-generation sequencing.