Laser capture microdissection microscopy and genome sequencing of the avian malaria parasite, Plasmodium relictum
Acquiring genomic material from avian malaria parasites for genome sequencing has proven problematic due to the nucleation of avian erythrocytes, which produces a large ratio of host to parasite DNA (∼1 million to 1 bp). We tested the ability of laser capture microdissection microscopy to isolate parasite cells from individual avian erythrocytes for four avian Plasmodium species, and subsequently applied whole genome amplification and Illumina sequencing methods to Plasmodium relictum (lineage pSGS1) to produce sequence reads of the P. relictum genome. We assembled ∼335 kbp of parasite DNA from this species, but were unable to completely avoid contamination by host DNA and other sources. However, it is clear that laser capture microdissection holds promise for the isolation of genomic material from haemosporidian parasites in intracellular life stages. In particular, laser capture microdissection may prove useful for isolating individual parasite species from co-infected hosts. Although not explicitly tested in this study, laser capture microdissection may also have important applications for isolation of rare parasite lineages and museum specimens for which no fresh material exists.
KeywordsAvian malaria Plasmodium genome laser capture microdissection microscopy
This project was completed with support from the Athena Fund, the Cornell Lab of Ornithology, and the Cornell College of Veterinary Medicine (Ithaca, NY, USA), as well as by the Negaunee Foundation at the Field Museum of Natural History (Chicago, IL, USA). Funding was also provided by the European Social Fund under the global grant measure (VPI-3.1.-ŠMM-07-K-01-047) to the Lithuania Research Council. We would like to thank Johanna Dela Cruz, Paulina Pavinski Bitar, and Peter Schweizer for their assistance with technical aspects of LCMM, DNA extraction, and genome sequencing, respectively. We also thank the Wellcome Trust Sanger Institute for access to data from an unpublished Plasmodium relictum genome (raw data available via NCBI Accession Number PRJEB2579).
Compliance with ethical standards
Experimental procedures for this study were approved by the Ethical Commission of the Baltic Laboratory Animal Science Association (Lithuania) and Lithuanian State Food and Veterinary Office (Ref. no. 2012/01/04-0221), Lithuania. Experimental procedures conducted in the USA complied with IACUC permit 17601 UC Davis.
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