Rapid whole genome optical mapping of Plasmodium falciparum
© Riley et al; licensee BioMed Central Ltd. 2011
Received: 1 June 2011
Accepted: 26 August 2011
Published: 26 August 2011
Immune evasion and drug resistance in malaria have been linked to chromosomal recombination and gene copy number variation (CNV). These events are ideally studied using comparative genomic analyses; however in malaria these analyses are not as common or thorough as in other infectious diseases, partly due to the difficulty in sequencing and assembling complete genome drafts. Recently, whole genome optical mapping has gained wide use in support of genomic sequence assembly and comparison. Here, a rapid technique for producing whole genome optical maps of Plasmodium falciparum is described and the results of mapping four genomes are presented.
Four laboratory strains of P. falciparum were analysed using the Argus™ optical mapping system to produce ordered restriction fragment maps of all 14 chromosomes in each genome. Plasmodium falciparum DNA was isolated directly from blood culture, visualized using the Argus™ system and assembled in a manner analogous to next generation sequence assembly into maps (AssemblyViewer™, OpGen Inc.®). Full coverage maps were generated for P. falciparum strains 3D7, FVO, D6 and C235. A reference P. falciparum in silico map was created by the digestion of the genomic sequence of P. falciparum with the restriction enzyme AflII, for comparisons to genomic optical maps. Maps were then compared using the MapSolver™ software.
Genomic variation was observed among the mapped strains, as well as between the map of the reference strain and the map derived from the putative sequence of that same strain. Duplications, deletions, insertions, inversions and misassemblies of sizes ranging from 3,500 base pairs up to 78,000 base pairs were observed. Many genomic events occurred in areas of known repetitive sequence or high copy number genes, including var gene clusters and rifin complexes.
This technique for optical mapping of multiple malaria genomes allows for whole genome comparison of multiple strains and can assist in identifying genetic variation and sequence contig assembly. New protocols and technology allowed us to produce high quality contigs spanning four P. falciparum genomes in six weeks for less than $1,000.00 per genome. This relatively low cost and quick turnaround makes the technique valuable compared to other genomic sequencing technologies for studying genetic variation in malaria.
Malaria is caused by various species of the genus Plasmodium, the most prevalent and deadly of which is Plasmodium falciparum[1, 2]. With ~40% of the world's population at risk for malaria, efforts in prevention, eradication and treatment of the disease are globally vital [2, 3]. Despite efforts to develop vaccines and drugs to combat malaria, vaccine escape and drug resistance continue to be a problem [1, 4, 5]. The ability to compare whole genomes could assist in these efforts, as genetic variation and recombination have been shown to facilitate antigen diversity, immune escape and evolution of anti-malarial drug resistance [4, 6–9]. Proposed mechanisms for these events include chromosome translocation and recombination, segmental duplication and CNV [10–13]. Ideally, fully sequenced and assembled genomes would be a way to study these mechanisms; however, this continues to be expensive and labour intensive [12–14]. Optical mapping provides an alternative to study these events, but to date has not been used for this purpose in malaria, partly due to the impracticality of scaling up previous mapping techniques to multiple genomes .
The 14 chromosome, 14 Mbp P. falciparum strain 3D7 genome was published in 2002, and to date this is the only complete, assembled genome sequence for this organism . Optical mapping of DNA was used to assist in assembly of this published genome sequence, but until now has not been widely used on any other species or strains [15, 17]. The Argus™ optical mapping system represents a radical advancement in whole genome optical mapping technology, and is being widely used for studies in bacterial identification, evolution, comparative genomics and genome assembly [18–22]. By developing new parasite isolation techniques and DNA extraction protocols, stated Argus™ limitations were surpassed to produce whole genome maps of malaria genomes for under $1,000.00 each, fully assembled in less than one week.
Parasite isolation and DNA extraction
DNA extraction was performed on this purified parasite sample according to the Gram-negative Bacterial DNA Preparation protocol available from Opgen, Inc.®. The parasites were lysed and resulting DNA was mixed with magnetic beads, subjected to a variety of wash buffers and eluted off the beads using wide bore pipette tips. Modifications to the manufacturer's protocol included addition of a 20 minute extension of initial lysis time, reduction in elution volume from 90 μL to 75 μL, and an extra two elutions at 65°C for 15 minutes each. Between 4 μL and 6 μL DNA was applied to mapcards (cards run on the Argus™) and run on the Argus™ system per manufacturer's specifications, after proper quality controls checks and appropriate dilutions using Opgen® QCards.
Mapset filter and contig assembly
Genome alignment and comparative genomics
Summary of Results
Total Length (Mb)
Variations to Referencea
Maximum Single Variationb
3D7 Optical Map
The major hurdle to optically mapping P. falciparum was sample preparation. Because the parasites can be grown only in blood culture, there is a possibility of contamination of the sample by human white blood cells or nucleated erythrocytes. As there is no amplification involved in this technique, even a single human cell (3,300 Mb DNA/cell) could overwhelm the sample with human DNA and mask the comparatively small 24 Mb/cell malaria genome. This was overcome using the parasite isolation technique described in the methods. The success of this method was such that no contigs in the assemblies failed to match a region of the sequenced Plasmodium genome.
Optical genomic mapping technology represents a significant advancement in comparative genomic research of malaria parasites. The optical maps of the four P. falciparum strains presented here demonstrate significant genome variation, much of which can be traced to regions known to contain coding sequence implicated in antigenic variation. This confirms findings generated via more difficult and expensive research methods that have suggested chromosomal translocations and segmental duplications are associated with immune escape and drug resistance in malaria [6–8, 10, 11]. Optical mapping resolves these types of genomic events and the techniques described in this paper allow for inexpensive, rapid, robust production of whole genome optical maps for malaria. This method also opens the door for assistance with genome assembly, as has been demonstrated with optical mapping of other large genomes [25–27]. The development of optical maps and fully assembled malaria genomes will increase knowledge of genetic variation in malaria parasites and thus enhance the ability to combat this disease.
We would like to thank Kathy Moch and Patty Lee for parasite culture, Charlotte Lanteri and Zachary Tycz for development and assistance with parasite isolation techniques, Edwin Kamau for advice on DNA preparation and Opgen®, Inc. for technical support with special thanks to Trevor Wagner for advice and assistance with DNA preparation and software manipulation and Louis Gardner and Emily Zentz for assistance with sequence assembly.
The opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of the Department of the Army or the Department of Defense.
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