title: Laser capture microdissection microscopy and genome sequencing of the avian malaria parasite, Plasmodium relictum.
abstract: 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.
authors.affiliation: Department of Ecology and Evolutionary Biology, College of Agricultural and Life Sciences, Cornell University, Ithaca, NY, 14853, USA. hlutz@fieldmuseum.org.|Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA. hlutz@fieldmuseum.org.|Cornell Lab of Ornithology, Cornell University, Ithaca, NY, 14853, USA. hlutz@fieldmuseum.org.|Integrative Research Center, The Field Museum of Natural History, Chicago, IL, 60605, USA. hlutz@fieldmuseum.org.
authors.forename: Holly L
authors.initials: HL
authors.lastname: Lutz
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Marra
authors.affiliation: Integrative Research Center, The Field Museum of Natural History, Chicago, IL, 60605, USA.
authors.forename: Felix
authors.initials: F
authors.lastname: Grewe
authors.affiliation: Department of Entomology, University of California, Davis, CA, 95616, USA.
authors.forename: Jenny S
authors.initials: JS
authors.lastname: Carlson
authors.affiliation: Nature Research Centre, Akademijos 2, Vilnius, LT-08412, Lithuania.
authors.forename: Vaidas
authors.initials: V
authors.lastname: Palinauskas
authors.affiliation: Nature Research Centre, Akademijos 2, Vilnius, LT-08412, Lithuania.
authors.forename: Gediminas
authors.initials: G
authors.lastname: Valkiūnas
authors.affiliation: Department of Population Medicine and Diagnostic Sciences, College of Veterinary Medicine, Cornell University, Ithaca, NY, 14853, USA. mjs297@cornell.edu.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Stanhope
mesh_terms: D000818:Animals; D001717:Birds; D018503:Genome, Protozoan; D061346:Laser Capture Microdissection; D008289:Malaria, Avian / Q000469:parasitology*; D010961:Plasmodium / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods
keywords: Avian malaria; Plasmodium genome; laser capture microdissection microscopy