Sequencing and annotation of mitochondrial genomes from individual parasitic helminths.
Mitochondrial (mt) genomics has significant implications in a range of fundamental areas of parasitology, including evolution, systematics, and population genetics as well as explorations of mt biochemistry, physiology, and function. Mt genomes also provide a rich source of markers to aid molecular epidemiological and ecological studies of key parasites. However, there is still a paucity of information on mt genomes for many metazoan organisms, particularly parasitic helminths, which has often related to challenges linked to sequencing from tiny amounts of material. The advent of next-generation sequencing (NGS) technologies has paved the way for low cost, high-throughput mt genomic research, but there have been obstacles, particularly in relation to post-sequencing assembly and analyses of large datasets. In this chapter, we describe protocols for the efficient amplification and sequencing of mt genomes from small portions of individual helminths, and highlight the utility of NGS platforms to expedite mt genomics. In addition, we recommend approaches for manual or semi-automated bioinformatic annotation and analyses to overcome the bioinformatic "bottleneck" to research in this area. Taken together, these approaches have demonstrated applicability to a range of parasites and provide prospects for using complete mt genomic sequence datasets for large-scale molecular systematic and epidemiological studies. In addition, these methods have broader utility and might be readily adapted to a range of other medium-sized molecular regions (i.e., 10-100 kb), including large genomic operons, and other organellar (e.g., plastid) and viral genomes.
Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Corner Flemington Road & Park Drive, Parkville, VIC, 3010, Australia, ajex@unimelb.edu.au.
Aaron R
AR
Jex
D Timothy
DT
Littlewood
Robin B
RB
Gasser
D000818:Animals; D004587:Electrophoresis, Agar Gel / Q000379:methods; D049751:Genome, Helminth; D054629:Genome, Mitochondrial; D023281:Genomics / Q000379:methods*; D006376:Helminths / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D058977:Molecular Sequence Annotation / Q000379:methods; D010271:Parasites / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods