Genomics and integrated systems biology in Plasmodium falciparum: a path to malaria control and eradication

Abstract

The first draft of the human malaria parasite's genome was released in 2002. Since then, the malaria scientific community has witnessed a steady embrace of new and powerful functional genomic studies. Over the years, these approaches have slowly revolutionized malaria research and enabled the comprehensive, unbiased investigation of various aspects of the parasite's biology. These genome-wide analyses delivered a refined annotation of the parasite's genome, delivered a better knowledge of its RNA, proteins and metabolite derivatives, and fostered the discovery of new vaccine and drug targets. Despite the positive impacts of these genomic studies, most research and investment still focus on protein targets, drugs and vaccine candidates that were known before the publication of the parasite genome sequence. However, recent access to next-generation sequencing technologies, along with an increased number of genome-wide applications, is expanding the impact of the parasite genome on biomedical research, contributing to a paradigm shift in research activities that may possibly lead to new optimized diagnosis and treatments. This review provides an update of Plasmodium falciparum genome sequences and an overview of the rapid development of genomics and system biology applications that have an immense potential of creating powerful tools for a successful malaria eradication campaign.

Figure 1. Methods for next-generation sequencing of the human malaria parasite's genome

Library preparation for sequencing on Illumina® NGS platforms usually involves the fragmentation of the dsDNA by a method of choice (e.g., sonication), end repair (fill in), and the addition of a single A tail on the 3’ end of each DNA strand before specific and oriented double-stranded adapters can be ligated. These chimera fragments are then PCR-amplified using specific primers that contain sequences compatible with the oligos attached to the Illumina® sequencing flowcell. In the amplification-free protocol (8), the PCR step is entirely skipped. The adapters are thus customized in order to contain the flowcell-compatible sequences in the absence of PCR. After adapter ligation, the library can be eventually size selected and purified prior to hybridization on the flowcell and sequencing. In both the optimized amplification (6) and the linear amplification protocols (7) the PCR step is performed with a polymerase that is different from the original Illumina® protocol. The linear amplification protocol uses the T7 polymerase and therefore requires that one of the adapters contains the sequence corresponding to the T7 promoter. In the case of the optimized amplification protocol, a mix of enzymes is used to perform the PCR (for example, a combination of the NEB Phusion and the Takara Ex Taq have been successfully used in the past (6,16)). The parameters for PCR cycling have also been adapted to the very high AT-content of the genome.

Figure 2. A system biology approach to understand the parasite biology for the design of new drug and vaccines strategies

Genomes to identify genetic diversity and drug resistances, Epigenomes to understand transcriptional regulation, Transcriptomes to evaluate mRNA steady state, Proteomes to evaluate protein levels, Immunomes to discovered protective antigens, Interactomes to comprehend protein-protein interactions, Metabolomes to evaluate metabolites.