Use of high-density tiling microarrays to identify mutations globally and elucidate mechanisms of drug resistance in Plasmodium falciparum

Abstract

Background: The identification of genetic changes that confer drug resistance or other phenotypic changes in pathogens can help optimize treatment strategies, support the development of new therapeutic agents, and provide information about the likely function of genes. Elucidating mechanisms of phenotypic drug resistance can also assist in identifying the mode of action of uncharacterized but potent antimalarial compounds identified in high-throughput chemical screening campaigns against Plasmodium falciparum.

Whole-genome sequencing and microarray analysis of ex vivo Plasmodium vivax reveal selective pressure on putative drug resistance genes

“…The algorithms designed for analysis of our P. falciparum microarray (24) were extended to analyze hybridizations of genomic DNA to the P. vivax array. Despite the lower probe density of 6-bp spacing compared with our P. falciparum array with 2-to 3-bp spacing, we observed clear signals for polymorphisms.…”

“…The appearance of SNPs in the genomic DNA of the patient-derived isolate compared with the reference strain resulted in hybridization intensities that were lower in the patient isolate than in the reference. The log of the ratio of intensities of the patient isolate IQ07 to the reference strain SalI was analyzed using a one-tailed z-test, and an F-test was used to predict the base-pair position of the polymorphism as described previously (24). We classified probes with a z-test p value of less than 1 × 10 −15 as highly likely to contain mutations in the isolate, resulting in a total of 8,118 predicted polymorphisms (Dataset S2).…”

“…The polymorphism detection was performed as previously described for P. falciparum (24). Briefly, using a sliding window of three overlapping probes, we scanned for sets that had significantly lower hybridization in IQ07 when compared with the SalI reference that were indicative of a polymorphism as determined by z-test using an empirically derived SD.…”

“…Here, we report whole-genome sequencing results with 30× coverage of a P. vivax isolate obtained directly ex vivo without further propagation in vitro or in monkeys. In addition to wholegenome sequencing, the parasite isolate was analyzed using a whole-genome tiling microarray (14,24). Our studies provide thousands of cross-validated polymorphisms in this Peruvian isolate of P. vivax, which are applicable to analysis of genetic diversity in P. vivax and to the identification of highly variable genes, such as a P. vivax multidrug resistance protein (pvmrp1, PVX_097025), which may be under immune or drug pressure.…”

Validation of isoleucine utilization targets in Plasmodium falciparum

“…This high coverage means that the majority of newly emerged SNPs in a drugresistant clone can be readily detected as a loss of signal at multiple consecutive probes covering the SNP. The probability of a genomic change is calculated from the number of consecutive probes showing a hybridization difference relative to the parental reference genome (10). The hybridizations of the two clones were compared with two 3D7 reference hybridizations using a P value cutoff of 1 × 10 −10 .…”