Plasmodium copy number variation scan: gene copy numbers evaluation in haploid genomes

Abstract

BackgroundIn eukaryotic genomes, deletion or amplification rates have been estimated to be a thousand more frequent than single nucleotide variation. In Plasmodium falciparum, relatively few transcription factors have been identified, and the regulation of transcription is seemingly largely influenced by gene amplification events. Thus copy number variation (CNV) is a major mechanism enabling parasite genomes to adapt to new environmental changes.MethodsCurrently, the detection of CNVs is based on quantitative PCR (qPCR), which is significantly limited by the relatively small number of genes that can be analysed at any one time. Technological advances that facilitate whole-genome sequencing, such as next generation sequencing (NGS) enable deeper analyses of the genomic variation to be performed. Because the characteristics of Plasmodium CNVs need special consideration in algorithms and strategies for which classical CNV detection programs are not suited a dedicated algorithm to detect CNVs across the entire exome of P. falciparum was developed. This algorithm is based on a custom read depth strategy through NGS data and called PlasmoCNVScan.ResultsThe analysis of CNV identification on three genes known to have different levels of amplification and which are located either in the nuclear, apicoplast or mitochondrial genomes is presented. The results are correlated with the qPCR experiments, usually used for identification of locus specific amplification/deletion.ConclusionsThis tool will facilitate the study of P. falciparum genomic adaptation in response to ecological changes: drug pressure, decreased transmission, reduction of the parasite population size (transition to pre-elimination endemic area).

Mitochondrial heteroplasmy is responsible for Atovaquone drug resistance in Plasmodium falciparum

“…The majority of mitochondrial copy numbers from different samples ranged from 10 to 30, suggesting significant copy number variation in mitochondria globally (F-test p-value < 2.2e-16). The results are in agreement with recent reports of qPCR experiments 31 which only used cytochrome b to estimate copy number.…”

A surrogate marker of piperaquine-resistant Plasmodium falciparum malaria: a phenotype–genotype association study

“… 1 Whole-genome sequencing was done with Illumina paired-reads sequencing. 1 Data were integrated into the Whole-genome Data Manager database 14 and exomes of piperaquine-resistant and piperaquine-sensitive lines were compared after excluding low-coverage positions (ie, lower than 25% of the genome-wide mean coverage). Genes from highly variable multigene families ( var, rifin, phist , and stevor ) were excluded.…”

“… 1 SNPs and CNVs were investigated using PlasmoCNVScan and the Phen2gen software ( appendix ). 14 Plasmepsin 2 and mdr1 copy number was determined by qPCR ( appendix ). Steady-state plasmepsin 2 mRNA concentrations were measured by RT-qPCR ( appendix ) and plasmepsin 2 protein expression by immunoblotting ( appendix ).…”

New directions in antimalarial target validation

“…These are dominated by genomic approaches which seek to either knock-in or out of a particular gene that encodes for an essential protein in the life cycle of the parasite [10]. Knockdown via conditional and inducible gene expression tools have also taken center stage in an effort to validate enzyme essentiality in Plasmodium, especially in instances where a complete knockout is undesirable [46]. Controlling protein activity at the RNA level with transcript interference and degradation tools has also led to a considerable improvement in target validation.…”

“…This cycle is repeated several times until the removal of all episomes and isolation of integrated forms (Figure 1(a)) [57]. Thanks to parasite haploidy [46], a single crossover event leads to knockout of parasites' target genes. But despite the advances brought by this method, it remains a long and inefficient procedure, with a timeframe of approximately 12 weeks required to generate the transgenic parasite [10].…”