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Development of a single nucleotide polymorphism barcode to genotype Plasmodium vivax infections.

Baniecki ML, Faust AL, Schaffner SF, Park DJ, Galinsky K, Daniels RF, Hamilton E, Ferreira MU, Karunaweera ND, Serre D, Zimmerman PA, Sá JM, Wellems TE, Musset L, Legrand E, Melnikov A, Neafsey DE, Volkman SK, Wirth DF, Sabeti PC - PLoS Negl Trop Dis (2015)

Bottom Line: We found that the P. vivax barcode is robust, as it requires only a small quantity of DNA (limit of detection 0.3 ng/μl) to yield reproducible genotype calls, and detects polymorphic genotypes with high sensitivity.Population genetic and statistical analyses show the barcode captures high degrees of population diversity and differentiates geographically distinct populations.Our 42-SNP barcode provides a robust, informative, and standardized genetic marker set that accurately identifies a genomic signature for P. vivax infections.

View Article: PubMed Central - PubMed

Affiliation: Broad Institute, Cambridge, Massachusetts, United States of America.

ABSTRACT
Plasmodium vivax, one of the five species of Plasmodium parasites that cause human malaria, is responsible for 25-40% of malaria cases worldwide. Malaria global elimination efforts will benefit from accurate and effective genotyping tools that will provide insight into the population genetics and diversity of this parasite. The recent sequencing of P. vivax isolates from South America, Africa, and Asia presents a new opportunity by uncovering thousands of novel single nucleotide polymorphisms (SNPs). Genotyping a selection of these SNPs provides a robust, low-cost method of identifying parasite infections through their unique genetic signature or barcode. Based on our experience in generating a SNP barcode for P. falciparum using High Resolution Melting (HRM), we have developed a similar tool for P. vivax. We selected globally polymorphic SNPs from available P. vivax genome sequence data that were located in putatively selectively neutral sites (i.e., intergenic, intronic, or 4-fold degenerate coding). From these candidate SNPs we defined a barcode consisting of 42 SNPs. We analyzed the performance of the 42-SNP barcode on 87 P. vivax clinical samples from parasite populations in South America (Brazil, French Guiana), Africa (Ethiopia) and Asia (Sri Lanka). We found that the P. vivax barcode is robust, as it requires only a small quantity of DNA (limit of detection 0.3 ng/μl) to yield reproducible genotype calls, and detects polymorphic genotypes with high sensitivity. The markers are informative across all clinical samples evaluated (average minor allele frequency > 0.1). Population genetic and statistical analyses show the barcode captures high degrees of population diversity and differentiates geographically distinct populations. Our 42-SNP barcode provides a robust, informative, and standardized genetic marker set that accurately identifies a genomic signature for P. vivax infections.

No MeSH data available.


Related in: MedlinePlus

WGA does not affect results in HRM analysis.The derivative melt graphs show the comparison of Tm profiles of a representative assay (14) tested with mixtures of monogenomic clinical samples processed with and without WGA prior to PCR. The ratios of reference (T) allele to alternate (C) allele in the mixtures were: (A) 1:2 and 1:4 and (B) 4:1 and 2:1.
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pntd.0003539.g002: WGA does not affect results in HRM analysis.The derivative melt graphs show the comparison of Tm profiles of a representative assay (14) tested with mixtures of monogenomic clinical samples processed with and without WGA prior to PCR. The ratios of reference (T) allele to alternate (C) allele in the mixtures were: (A) 1:2 and 1:4 and (B) 4:1 and 2:1.

Mentions: Using a series of known allelic mixtures, we found that WGA prior to PCR did not affect the genotyping accuracy of the HRM assays or resulting genotype. We selected assays representing each SNP type present in the barcode (CT, CA, GT, and GA). We then evaluated mixtures of monogenomic clinical samples with varying ratios of reference and alternate alleles (1:1, 1:2, 1:4, 2:1 and 4:1). The HRM analysis showed that all assay SNP types detected the ratio of reference and alternate alleles with the same accuracy in duplicate in sample mixtures processed with and without WGA (Fig. 2). We further compared the 42 assays using 17 clinical samples from French Guiana with and without WGA, and found no significant differences. For all 42 assays and 17 samples, all genotype calls were concordant between sample treatments.


Development of a single nucleotide polymorphism barcode to genotype Plasmodium vivax infections.

Baniecki ML, Faust AL, Schaffner SF, Park DJ, Galinsky K, Daniels RF, Hamilton E, Ferreira MU, Karunaweera ND, Serre D, Zimmerman PA, Sá JM, Wellems TE, Musset L, Legrand E, Melnikov A, Neafsey DE, Volkman SK, Wirth DF, Sabeti PC - PLoS Negl Trop Dis (2015)

WGA does not affect results in HRM analysis.The derivative melt graphs show the comparison of Tm profiles of a representative assay (14) tested with mixtures of monogenomic clinical samples processed with and without WGA prior to PCR. The ratios of reference (T) allele to alternate (C) allele in the mixtures were: (A) 1:2 and 1:4 and (B) 4:1 and 2:1.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4362761&req=5

pntd.0003539.g002: WGA does not affect results in HRM analysis.The derivative melt graphs show the comparison of Tm profiles of a representative assay (14) tested with mixtures of monogenomic clinical samples processed with and without WGA prior to PCR. The ratios of reference (T) allele to alternate (C) allele in the mixtures were: (A) 1:2 and 1:4 and (B) 4:1 and 2:1.
Mentions: Using a series of known allelic mixtures, we found that WGA prior to PCR did not affect the genotyping accuracy of the HRM assays or resulting genotype. We selected assays representing each SNP type present in the barcode (CT, CA, GT, and GA). We then evaluated mixtures of monogenomic clinical samples with varying ratios of reference and alternate alleles (1:1, 1:2, 1:4, 2:1 and 4:1). The HRM analysis showed that all assay SNP types detected the ratio of reference and alternate alleles with the same accuracy in duplicate in sample mixtures processed with and without WGA (Fig. 2). We further compared the 42 assays using 17 clinical samples from French Guiana with and without WGA, and found no significant differences. For all 42 assays and 17 samples, all genotype calls were concordant between sample treatments.

Bottom Line: We found that the P. vivax barcode is robust, as it requires only a small quantity of DNA (limit of detection 0.3 ng/μl) to yield reproducible genotype calls, and detects polymorphic genotypes with high sensitivity.Population genetic and statistical analyses show the barcode captures high degrees of population diversity and differentiates geographically distinct populations.Our 42-SNP barcode provides a robust, informative, and standardized genetic marker set that accurately identifies a genomic signature for P. vivax infections.

View Article: PubMed Central - PubMed

Affiliation: Broad Institute, Cambridge, Massachusetts, United States of America.

ABSTRACT
Plasmodium vivax, one of the five species of Plasmodium parasites that cause human malaria, is responsible for 25-40% of malaria cases worldwide. Malaria global elimination efforts will benefit from accurate and effective genotyping tools that will provide insight into the population genetics and diversity of this parasite. The recent sequencing of P. vivax isolates from South America, Africa, and Asia presents a new opportunity by uncovering thousands of novel single nucleotide polymorphisms (SNPs). Genotyping a selection of these SNPs provides a robust, low-cost method of identifying parasite infections through their unique genetic signature or barcode. Based on our experience in generating a SNP barcode for P. falciparum using High Resolution Melting (HRM), we have developed a similar tool for P. vivax. We selected globally polymorphic SNPs from available P. vivax genome sequence data that were located in putatively selectively neutral sites (i.e., intergenic, intronic, or 4-fold degenerate coding). From these candidate SNPs we defined a barcode consisting of 42 SNPs. We analyzed the performance of the 42-SNP barcode on 87 P. vivax clinical samples from parasite populations in South America (Brazil, French Guiana), Africa (Ethiopia) and Asia (Sri Lanka). We found that the P. vivax barcode is robust, as it requires only a small quantity of DNA (limit of detection 0.3 ng/μl) to yield reproducible genotype calls, and detects polymorphic genotypes with high sensitivity. The markers are informative across all clinical samples evaluated (average minor allele frequency > 0.1). Population genetic and statistical analyses show the barcode captures high degrees of population diversity and differentiates geographically distinct populations. Our 42-SNP barcode provides a robust, informative, and standardized genetic marker set that accurately identifies a genomic signature for P. vivax infections.

No MeSH data available.


Related in: MedlinePlus