开发扩增子深度测序标记和数据分析管道，用于对多克隆疟疾感染进行基因分型。
Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections.
机构信息
Swiss Tropical and Public Health Institute, Basel, Switzerland.
University of Basel, Basel, Switzerland.
出版信息
BMC Genomics. 2017 Nov 13;18(1):864. doi: 10.1186/s12864-017-4260-y.
BACKGROUND
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.
METHODS
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.
RESULTS
Cpmp was highly diverse (H = 0.96) in contrast to csp (H = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold.
CONCLUSIONS
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.
背景
扩增子深度测序可灵敏检测少数克隆体，并提高多克隆疟原虫感染的基因分型辨别能力。在疟疾流行学研究和疗效试验中，需要新的扩增子测序和数据分析方案来进行基因分型。
方法
对寄生虫培养株混合物和 37 个现场样本进行分子标记 csp 和新型标记 cpmp 的靶向测序，应用允许在单次测序运行中对多达 384 个样本进行多重化的方案。开发了软件“haplotypR”用于数据分析。
结果
Cpmp 高度多样化（H=0.96），而 csp 则多样化程度较低（H=0.57）。如果频率>1%，则可以可靠地检测到少数克隆体。低于该阈值时，会观察到由于测序错误导致的假单倍型调用。
结论
为了可靠地检测到极低频率的单倍型，最好进行重复实验，并且应争取每个扩增子覆盖>10,000 个读数。与长度多态性标记 msp2 相比，高度多重化的扩增子测序在检测少数克隆体方面显示出更高的灵敏度。
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