Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics

Suo, Wei; Shi, Xiaowen; Xu, Sha; Xiao, Li; Yang, Lin

doi:10.1371/journal.pone.0230445

Cited by 11 publications

(10 citation statements)

References 16 publications

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“…This requirement increases the cost and limits the number of service providers available. GRR technologies can be improved, and new platforms are still being developed (11)(12)(13)(14)(15). It would be ideal to have open, patent-unencumbered technologies that could be easily used by any molecular biology laboratory.…”

Section: Consequently Several Genomic Reduced Representation (Grr) Tmentioning

confidence: 99%

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

Ziarsolo

Hasing

Hilario

et al. 2020

Preprint

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K-seq, a new genotyping methodology based on the amplification of genomic regions using two steps of Klenow amplification with short oligonucleotides, followed by standard PCR and Illumina sequencing, is presented. The protocol was accompanied by software developed to aid with primer set design. As the first examples, K-seq in species as diverse as tomato, dog and wheat was developed. K-seq provided genetic distances similar to those based on WGS in dogs. Experiments comparing K-seq and GBS in tomato showed similar genetic results, although K-seq had the advantage of finding more SNPs for the same number of Illumina reads. The technology reproducibility was tested with two independent runs of the tomato samples, and the correlation coefficient of the SNP coverages between samples was 0.8 and the genotype match was above 94%. K-seq also proved to be useful in polyploid species. The wheat samples generated specific markers for all subgenomes, and the SNPs generated from the diploid ancestors were located in the expected subgenome with accuracies greater than 80%. K-seq is an open, patent-unencumbered, easy-to-set-up, cost-effective and reliable technology ready to be used by any molecular biology laboratory without special equipment in many genetic studies.

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Section: Consequently Several Genomic Reduced Representation (Grr) Tmentioning

confidence: 99%

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

Ziarsolo

Hasing

Hilario

et al. 2020

Preprint

View full text Add to dashboard Cite

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“…The KASP method introduces fluorescence resonance energy transfer (FRET) for signal generation, where two fluorescent cassettes are used for the identification of allele-specific amplification for a single bi-allelic SNP [ 9 ]. The process begins with the first round of the PCR, where one of the allele-specific primers matches the target SNP and, with the common reverse primer, amplifies the target region.…”

Section: Introductionmentioning

confidence: 99%

“…This method has been used, for example, for the detection of SNPs associated with the efficacy of specific drugs [ 9 ]; for genotyping candidate genes associated with the development of genetic diseases, such as Huntington’s disease [ 10 ]; or for the genotyping of SNPs associated with G6PD deficiency [ 11 ].…”

Section: Introductionmentioning

confidence: 99%

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

Alvarez-Fernandez

Bernal

Fradejas

et al. 2021

Malar J

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Background The emergence and spread of anti-malarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known anti-malarials. This anti-malarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorphisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programmes. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. Methods Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. Results The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed. Conclusions The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

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“…This method has been used, for example, for the detection of SNPs associated with e cacy of speci c drugs [9]; for genotyping candidate genes associated with the development of genetic diseases, such as Hungtinton's disease [10]; or for the genotyping of SNPs associated with G6PD de ciency [11].…”

Section: Introductionmentioning

confidence: 99%

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

Alvarez-Fernandez

Bernal

Villajos

et al. 2020

Preprint

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Background The emergence and spread of antimalarial resistance continues to hinder malaria control. Plasmodium falciparum, the species that causes most human malaria cases and most deaths, has shown resistance to almost all known antimalarials. This antimalarial resistance arises from the development and subsequent expansion of Single Nucleotide Polymorfisms (SNPs) in specific parasite genes. A quick and cheap tool for the detection of drug resistance can be crucial and very useful for use in hospitals and in malaria control programs. It has been demonstrated in different contexts that genotyping by Kompetitive Allele Specific PCR (KASP), is a simple, fast and economical method that allows a high-precision biallelic characterization of SNPs, hence its possible utility in the study of resistance in P. falciparum. Methods Three SNPs involved in most cases of resistance to the most widespread antimalarial treatments have been analyzed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analyzed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method. Results The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analyzed. Conclusions The KASP assays developed in our study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.

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Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics

Cited by 11 publications

References 16 publications

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

K-seq, an affordable, reliable, and open Klenow NGS-based genotyping technology

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

KASP: a genotyping method to rapid identification of resistance in Plasmodium falciparum

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