Sequencing and Annotation of Mitochondrial Genomes from Individual Parasitic Helminths

  • Aaron R. Jex
  • D. Timothy Littlewood
  • Robin B. GasserEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1201)


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.


Parasitic Helminth Complete Mitochondrial Genome Molecular Size Marker Ethylenediaminetetraacetic Acid Disodium Salt Individual Nematode 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Our research has been supported largely through grants from the Australian Research Council (ARC) and the National Health and Medical Research Council. Other support from the Alexander von Humboldt Foundation, Australian Academy of Science, the Fulbright Commission, Melbourne Water Corporation, the Victorian Life Sciences Computation Initiative (VLSCI), and the IBM Collaboratory is gratefully acknowledged.


  1. 1.
    Hu M, Gasser RB (2006) Mitochondrial genomes of parasitic nematodes—progress and perspectives. Trends Parasitol 22:78–84CrossRefPubMedGoogle Scholar
  2. 2.
    Jex AR, Littlewood DT, Gasser RB (2010) Toward next-generation sequencing of mitochondrial genomes—focus on parasitic worms of animals and biotechnological implications. Biotechnol Adv 28:151–159CrossRefPubMedGoogle Scholar
  3. 3.
    Boore JL, Macey JR, Medina M (2005) Sequencing and comparing whole mitochondrial genomes of animals. In: Zimmer EA, Roalson X (eds) Molecular evolution: producing the biochemical data, part B. Elsevier, BurlingtonGoogle Scholar
  4. 4.
    Burger G, Lavrov DV, Forget L, Lang BF (2007) Sequencing complete mitochondrial and plastid genomes. Nat Protoc 2:603–614CrossRefPubMedGoogle Scholar
  5. 5.
    Hu M, Chilton NB, Gasser RB (2004) The mitochondrial genomics of parasitic nematodes of socio-economic importance: recent progress, and implications for population genetics and systematics. Adv Parasitol 56:133–212CrossRefPubMedGoogle Scholar
  6. 6.
    Lang BF, Burger G (2007) Purification of mitochondrial and plastid DNA. Nat Protoc 2:652–660CrossRefPubMedGoogle Scholar
  7. 7.
    Lavrov DV, Brown WM, Boore JL (2000) A novel type of RNA editing occurs in the mitochondrial tRNAs of the centipede Lithbius forficatus. Proc Natl Acad Sci U S A 97:13738–13742CrossRefPubMedCentralPubMedGoogle Scholar
  8. 8.
    Le TH, Blair D, McManus DP (2000) Mitochondrial genomes of human helminths and their use as markers in population genetics and phylogeny. Acta Trop 77:243–256CrossRefPubMedGoogle Scholar
  9. 9.
    Simison WB, Lindberg DR, Boore JL (2006) Rolling circle amplification of metazoan mitochondrial genomes. Mol Phylogenet Evol 39:562–567CrossRefPubMedGoogle Scholar
  10. 10.
    Gasser RB (2006) Molecular tools–advances, opportunities and prospects. Vet Parasitol 136:69–89CrossRefPubMedGoogle Scholar
  11. 11.
    Hu M, Jex AR, Campbell BE, Gasser RB (2007) Long PCR amplification of the entire mitochondrial genome from individual helminths for direct sequencing. Nat Protoc 2:2339–2344CrossRefPubMedGoogle Scholar
  12. 12.
    Hu M, Chilton NB, Gasser RB (2003) The mitochondrial genome of Strongyloides stercoralis (Nematoda)—idiosyncratic gene order and evolutionary implications. Int J Parasitol 33:1393–1408CrossRefPubMedGoogle Scholar
  13. 13.
    Mardis ER (2008) Next-generation DNA sequencing methods. Annu Rev Genomics Hum Genet 9:387–402CrossRefPubMedGoogle Scholar
  14. 14.
    Jex AR, Hall RS, Littlewood DT, Gasser RB (2010) An integrated pipeline for next-generation sequencing and annotation of mitochondrial genomes. Nucleic Acids Res 38:522–533CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Jex AR, Hu M, Littlewood DT, Waeschenbach A, Gasser RB (2008) Using 454 technology for long-PCR based sequencing of the complete mitochondrial genome from single Haemonchus contortus (Nematoda). BMC Genomics 9:11CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Jex AR, Waeschenbach A, Hu M, van Wyk JA, Beveridge I, Littlewood DT et al (2009) The mitochondrial genomes of Ancylostoma caninum and Bunostomum phlebotomum–two hookworms of animal health and zoonotic importance. BMC Genomics 10:79CrossRefPubMedCentralPubMedGoogle Scholar
  17. 17.
    Binladen J, Gilbert MT, Bollback JP, Panitz F, Bendixen C, Nielsen R et al (2007) The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS One 2:e197CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Glenn TC (2011) Field guide to next-generation DNA sequencers. Mol Ecol Resour 11:759–769CrossRefPubMedGoogle Scholar
  19. 19.
    Horner DS, Pavesi G, Castrignano T, De Meo PD, Liuni S, Sammeth M et al (2010) Bioinformatics approaches for genomics and post genomics applications of next-generation sequencing. Brief Bioinform 11:181–197CrossRefPubMedGoogle Scholar
  20. 20.
    Schattner P, Brooks AN, Lowe TM (2005) The tRNAscan-SE, snoscan and snoGPS web servers for the detection of tRNAs and snoRNAs. Nucleic Acids Res 33:W686–W689CrossRefPubMedCentralPubMedGoogle Scholar
  21. 21.
    Zhang DX, Hewitt GM (1996) Nuclear integrations: challenges for mitochondrial DNA markers. Trends Ecol Evol 11:247–251CrossRefPubMedGoogle Scholar
  22. 22.
    Keddie EM, Higazi T, Unnasch TR (1998) The mitochondrial genome of Onchocerca volvulus: sequence, structure and phylogenetic analysis. Mol Biochem Parasitol 95:111–127CrossRefPubMedGoogle Scholar
  23. 23.
    Lavrov DV, Brown WM (2001) Trichinella spiralis mtDNA: a nematode mitochondrial genome that encodes a putative ATP8 and normally structured tRNAS and has a gene arrangement relatable to those of coelomate metazoans. Genetics 157:621–637PubMedCentralPubMedGoogle Scholar
  24. 24.
    Okimoto R, Macfarlane JL, Clary DO, Wolstenholme DR (1992) The mitochondrial genomes of two nematodes, Caenorhabditis elegans and Ascaris suum. Genetics 130:471–498PubMedCentralPubMedGoogle Scholar
  25. 25.
    Nelson WS, Prodöhl PA, Avise JC (1996) Development and application of long-PCR for the assay of full-length animal mitochondrial DNA. Mol Ecol 5:807–810CrossRefPubMedGoogle Scholar
  26. 26.
    Chevreux B, Pfisterer T, Drescher B, Driesel AJ, Muller WE, Wetter T et al (2004) Using the miraEST assembler for reliable and automated mRNA transcript assembly and SNP detection in sequenced ESTs. Genome Res 14:1147–1159CrossRefPubMedCentralPubMedGoogle Scholar
  27. 27.
    Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18:821–829CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Zerbino DR (2010) Using the Velvet de novo assembler for short-read sequencing technologies. Curr Protoc Bioinformatics Chapter 11:Unit 11.15Google Scholar
  29. 29.
    Li Y, Hu Y, Bolund L, Wang J (2010) State of the art de novo assembly of human genomes from massively parallel sequencing data. Hum Genomics 4:271–277CrossRefPubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Aaron R. Jex
    • 1
  • D. Timothy Littlewood
    • 2
  • Robin B. Gasser
    • 1
    Email author
  1. 1.Faculty of Veterinary and Agricultural SciencesThe University of MelbourneParkvilleAustralia
  2. 2.Department of Life SciencesNatural History MuseumLondonUK

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