Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections

BMC Genomics

Methdology Article

Inoni Betuela¹  Johanna H. Kattenberg²  Camilla Messerli³  Ingrid Felger³  Natalie E. Hofmann³  Anita Lerch⁴  Stephen Wilcox⁵  Liam O’Connor⁵  Cristian Koepfli⁵  Ivo Mueller⁶ 

[1] Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea;Papua New Guinea Institute of Medical Research, Madang, Papua New Guinea;Present Address: Institute of Tropical Medicine, Antwerp, Belgium;Swiss Tropical and Public Health Institute, Basel, Switzerland;University of Basel, Basel, Switzerland;Swiss Tropical and Public Health Institute, Basel, Switzerland;University of Basel, Basel, Switzerland;Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia;Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia;University of Melbourne, Parkville, Australia;Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia;University of Melbourne, Parkville, Australia;Present Address: Institut Pasteur, Paris, France;

关键词: Plasmodium falciparum;    Malaria;    Amplicon sequencing;    SNP;    Haplotype clustering;    Multi-clone infections;    msp2;    csp;    cpmp;    HaplotypR software;   

DOI  :  10.1186/s12864-017-4260-y

 received in 2017-04-26, accepted in 2017-11-01,  发布年份 2017

来源: Springer

【 摘 要 】

BackgroundAmplicon 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.MethodsTargeted 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.ResultsCpmp 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.ConclusionsTo 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.

【 授权许可】

CC BY   
© The Author(s). 2017