A general SNP-based molecular barcode for Plasmodium falciparum identification and tracking

Abstract

Background: Single nucleotide polymorphism (SNP) genotyping provides the means to develop a practical, rapid, inexpensive assay that will uniquely identify any Plasmodium falciparum parasite using a small amount of DNA. Such an assay could be used to distinguish recrudescence from re-infection in drug trials, to monitor the frequency and distribution of specific parasites in a patient population undergoing drug treatment or vaccine challenge, or for tracking samples and determining purity of isolates in the laboratory during culture adaptation and sub-cloning, as well as routine passage.

Methods: A panel of twenty-four SNP markers has been identified that exhibit a high minor allele frequency (average MAF > 35%), for which robust TaqMan genotyping assays were constructed. All SNPs were identified through whole genome sequencing and MAF was estimated through Affymetrix array-based genotyping of a worldwide collection of parasites. These assays create a "molecular barcode" to uniquely identify a parasite genome.

Results: Using 24 such markers no two parasites known to be of independent origin have yet been found to have the same allele signature. The TaqMan genotyping assays can be performed on a variety of samples including cultured parasites, frozen whole blood, or whole blood spotted onto filter paper with a success rate > 99%. Less than 5 ng of parasite DNA is needed to complete a panel of 24 markers. The ability of this SNP panel to detect and identify parasites was compared to the standard molecular methods, MSP-1 and MSP-2 typing.

Conclusion: This work provides a facile field-deployable genotyping tool that can be used without special skills with standard lab equipment, and at reasonable cost that will unambiguously identify and track P. falciparum parasites both from patient samples and in the laboratory.

Figure 1

Distribution of single nucleotide polymorphisms assayed and resulting molecular bar codes for sequenced parasites. Twenty-four SNPs with an average minor allele frequency of at least 35% that were unlinked and assayable by TaqMan technology were selected from SNPs identified across the P. falciparum genome from sequencing efforts. The positions of the SNPs on the 14 chromosomes of P. falciparum are shown in A, with the sequence of the major and minor allele for each of the SNPs that comprise the molecular bar code shown in B. The position numbers in B (1 to 24) correspond to the positions in A, beginning with chromosome 1 through chromosome 14 and on each chromosome starting with the lowest coordinate number [13] and proceeding to the highest coordinate number on that chromosome (See Additional File 10). The major allele is indicated by a gray box and the minor allele by a white box. The yellow box (with an X) indicates no amplification. In all cases the TaqMan assay results matched sequence information.

Figure 2

TaqMan assays discriminate between the major and minor allele. Two representative assays (corresponding to 07_000490877 and 10_001403751, with the remaining assays found in Additional Files 5 and 6) run for a subset of the parasites (corresponding to a typical running of the assay) are shown, indicating the clear separation between the signal derived from the major and minor allele. The major allele (Allele X) is displayed on the X axis and the minor allele (Allele Y) on the Y axis for 20 independent strains along with a non template control (NTC) containing only water.

Figure 3

TaqMan assays quantify alleles within DNA mixtures. Relative amounts of each allele within a mixture of known amounts of indicated DNA were determined to correspond to the quantity of input DNA in the mixture. The relative ratios of Dd2:HB3 shown as green dots in panel A; and ratios for Dd2:3D7 and VS/1:HB3 shown on the X-axis for panels B and C, respectively. The major (red dots) and minor allele (blue dots) calls for 32 pure parasite DNA samples which type with either one or the other allele are indicated in Panel A. Panels B and C show the CT (threshold cycle) for the relative ratios of the indicated DNA samples for the 06_000145472 and 13_000158614 assays respectively. The closed squares indicate amplification of the allele labeled with VIC^® dye and the open triangles indicate amplification of the allele labeled with FAM™.

Figure 4

Comparison of MSP-1 and MSP-2 genotype with molecular bar code assay. Patient samples from Senegal and Malawi were genotyped using standard MSP1/2 methods. Examples of samples where both molecular bar code and MSP1/2 methods matched are shown in the upper right (single genome, white background) and the lower left (mixed genomes, orange background), with unmatched examples shown in the upper right and lower left (gray background). Samples having a single product for each of MSP-1 and MSP-2 were shown to have a single bar code (top left, white background) and a mixed bar code (top right, gray background). Mixed MSP-1 and MSP-2 samples were shown to have a single bar code (bottom left, gray background) and a mixed bar code (bottom right, orange background) using the molecular bar code technology.