Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections.
Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections.
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DOI：
10.1186/s12864-017-4260-y
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发表时间：
2017-11-13
期刊：
影响因子：
4.4
通讯作者：
Felger I
中科院分区：
文献类型：
--
作者：
Lerch A;Koepfli C;Hofmann NE;Messerli C;Wilcox S;Kattenberg JH;Betuela I;O'Connor L;Mueller I;Felger I
扩增子深度测序允许少数克隆的灵敏检测，并提高了对多克隆恶性疟原虫感染进行基因分型的区分能力。在恶性疟原虫的流行病学研究和药物疗效试验中，需要新的扩增子测序和数据分析方案进行基因分型。分子标记csp和新标记cpmp的靶向测序在寄生虫培养菌株和37个现场样品的混合物上进行，一式两份。应用了允许在单次测序运行中多路复用多达384个样品的方案。开发了用于数据分析的软件“HaplotypR”。 Cpmp是高度多样化的（He = 0.96），而CSP（He = 0.57）。如果少数克隆的频率> 1%，则稳健地检测到它们。在低于该阈值的情况下观察到由于测序错误导致的假单倍型判定。为了在非常低的频率下可靠地检测单倍型，实验最好一式两份进行，并且应该以>10 '000读数/扩增子的覆盖率为目标。与长度多态性标记MSP 2相比，高度多重扩增子测序在检测少数克隆中显示出更高的灵敏度。本文的在线版本（10.1186/s12864-017-4260-y）包含补充材料，可供授权用户使用。
Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum. Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software “HaplotypR” was developed for data analysis. Cpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10′000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones. The online version of this article (10.1186/s12864-017-4260-y) contains supplementary material, which is available to authorized users.
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