Abstract
BackgroundKnowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum) genome is crucial for understanding its evolution; however the 81% (A+T) genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX) high throughput sequencing technology for both whole genome sequencing and fine-resolution characterization of genetic exchange in malaria parasites.ResultsWe present a scheme to survey recombination in the haploid stage genomes of two sibling parasite clones, using whole genome pyrosequencing that includes a sliding window approach to predict recombination breakpoints. Whole genome shotgun (WGS) sequencing generated approximately 2 million reads, with an average read length of approximately 300 bp. De novo assembly using a combination of WGS and 3 kb paired end libraries resulted in contigs ≤ 34 kb. More than 8,000 of the 24,599 SNP markers identified between parents were genotyped in the progeny, resulting in a marker density of approximately 1 marker/3.3 kb and allowing for the detection of previously unrecognized crossovers (COs) and many non crossover (NCO) gene conversions throughout the genome.ConclusionsBy sequencing the 23 Mb genomes of two haploid progeny clones derived from a genetic cross at more than 30× coverage, we captured high resolution information on COs, NCOs and genetic variation within the progeny genomes. This study is the first to resequence progeny clones to examine fine structure of COs and NCOs in malaria parasites.
Highlights
Knowledge of the origins, distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum) genome is crucial for understanding its evolution; the 81% (A+T) genome poses challenges to high-throughput sequencing technologies
We show that Whole genome shotgun (WGS) sequencing using the 454 GS
Genome Sequencer FLX (FLX) sequencing platform is suitable for single nucleotide polymorphism (SNP) allele detection even in the highly (A+T) biased malaria parasite, P. falciparum
Summary
Distribution, and inheritance of variation in the malaria parasite (Plasmodium falciparum) genome is crucial for understanding its evolution; the 81% (A+T) genome poses challenges to high-throughput sequencing technologies. We explore the viability of the Roche 454 Genome Sequencer FLX (GS FLX) high throughput sequencing technology for both whole genome sequencing and fineresolution characterization of genetic exchange in malaria parasites. Advances in genotyping technology led to an explosion of studies to identify genes of interest using classical genetic approaches [1]. Such studies facilitate the discovery of genetic factors related to disease, drug resistance and environmental response. The ambiguous length of homopolymer runs, a primary limitation of this pyrosequencing-based method, may prohibit the sequencing of highly biased genomes
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