High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology.
High throughput resistance profiling of Plasmodium falciparum infections based on custom dual indexing and Illumina next generation sequencing-technology.
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DOI：
10.1038/s41598-017-02724-x
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发表时间：
2017-05-25
影响因子：
4.6
通讯作者：
Alifrangis M
中科院分区：
文献类型：
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作者：
Nag S;Dalgaard MD;Kofoed PE;Ursing J;Crespo M;Andersen LO;Aarestrup FM;Lund O;Alifrangis M
恶性疟原虫的遗传多态性可用于表明该寄生虫对抗疟药物的易感性及其地理来源。这两个因素都是监测抗疟药耐药性发展和传播的关键。在这项研究中，我们结合多重PCR、定制双标引和Miseq测序，对来自几内亚比绍的457例疟疾感染进行了高通量snp分析，每个样本的成本为10美元。通过对15个基因片段进行扩增和测序，我们覆盖了pfcrt、pfmdr1、pfdhfr、pfdhps以及pfK13的整个长度以及寄生虫起源的线粒体条形码中的20个耐药snp。对感兴趣的snp进行平均深度2043 reads的测序，在89.8-100%的样本中，对p值低于0.05的各个snp位置进行碱基调用。SNP数据表明，在几内亚比绍研究地区，pfK13中不存在具有青蒿素耐药性的SNP，而pfmdr186 N等位基因的患病率很高。线粒体条形码是一致的，并且适应了寄生虫的西非起源。有了这种方法，非常可靠的高通量抗疟药耐药性监测变得比以往任何时候都更容易负担。
Genetic polymorphisms in P. falciparum can be used to indicate the parasite’s susceptibility to antimalarial drugs as well as its geographical origin. Both of these factors are key to monitoring development and spread of antimalarial drug resistance. In this study, we combine multiplex PCR, custom designed dual indexing and Miseq sequencing for high throughput SNP-profiling of 457 malaria infections from Guinea-Bissau, at the cost of 10 USD per sample. By amplifying and sequencing 15 genetic fragments, we cover 20 resistance-conferring SNPs occurring in pfcrt, pfmdr1, pfdhfr, pfdhps, as well as the entire length of pfK13, and the mitochondrial barcode for parasite origin. SNPs of interest were sequenced with an average depth of 2,043 reads, and bases were called for the various SNP-positions with a p-value below 0.05, for 89.8–100% of samples. The SNP data indicates that artemisinin resistance-conferring SNPs in pfK13 are absent from the studied area of Guinea-Bissau, while the pfmdr1 86 N allele is found at a high prevalence. The mitochondrial barcodes are unanimous and accommodate a West African origin of the parasites. With this method, very reliable high throughput surveillance of antimalarial drug resistance becomes more affordable than ever before.