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

Abstract

29 30Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power 31 for genotyping multi-clone Plasmodium falciparum infections. Such high resolution is needed for molecular 32 monitoring of drug efficacy trials. Targeted sequencing of molecular marker csp and novel marker cpmp was 33 conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol to multiplex up 34 to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data 35 analysis. Cpmp was highly diverse (H e =0.96) in contrast to csp (H e =0.57). Minority clones were robustly 36 detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below 37 that threshold. To reliably detect haplotypes at very low frequencies, experiments are best performed in 38 duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic 39 marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority 40 clones. 41 42

The use of spatial and genetic tools to assess Plasmodium falciparum transmission in Lusaka, Zambia between 2011 and 2015

“…However, due to the high MOI and high importation, with the majority of infections likely acquired across a large area of Zambia, it was not possible to perform genetic spatial analyses with the SNP-barcode methods. Future work aiming to examine spatial genetics as potential tools for assessing the epidemiology of malaria parasites should consider genetic barcoding methods such as amplicon deep sequencing which may allow analysis of polyclonal samples [59]. Otherwise, researchers should seek areas of low transmission where imported cases are relatively few.…”

Microgeographic Epidemiology of Malaria Parasites in an Irrigated Area of Western Kenya by Deep Amplicon Sequencing

“…PCR-based genotyping, such as by size-polymorphic antigens or microsatellites, often underestimates MOI, as it is challenging to detect minority strains, which result in faint bands or weak fluorescent signals [13]. In contrast, deep amplicon sequencing has been shown to be more sensitive for detecting MOI [14][15][16]. Moreover, choice of molecular marker for amplicon sequencing, which can vary in terms of the number of single nucleotide polymorphisms (SNPs), can also impact the ability to assess MOI [14,16], and thus potentially the sensitivity to assess differences in MOI between varying transmission intensities.…”

“…In contrast, deep amplicon sequencing has been shown to be more sensitive for detecting MOI [14][15][16]. Moreover, choice of molecular marker for amplicon sequencing, which can vary in terms of the number of single nucleotide polymorphisms (SNPs), can also impact the ability to assess MOI [14,16], and thus potentially the sensitivity to assess differences in MOI between varying transmission intensities. Recently, Lerch et al [17] demonstrated that cpmp (PF3D7_0104100, "conserved Plasmodium membrane protein") detected a higher mean MOI and contained more SNPs than common molecular markers within parasites originating from Papua New Guinea.…”

“…Amplicon deep sequencing. Amplification and sequencing of the molecular markers ama1, cpmp, csp, and msp1 were performed based on previously published primers and protocols [14,16,17,22,23] with modifications. Briefly, the sequencing library was generated by three rounds of PCR.…”

Molecular Epidemiology of Plasmodium Falciparum by Multiplexed Amplicon Deep Sequencing in Senegal

“…Advances in next generation sequencing (NGS) technologies and bioinformatic analysis has enabled the accurate detection of clones and even minor clones, overcoming the limitations of these other methods [24]. These techniques provide a more accurate estimation of MOI compared to the standard PCR-based methods [25][26] and accurate assessment of genetic diversity in P. falciparum populations [27]. Indeed, using amplicon deep sequencing of Pfmsp1 using Illumina MiSeq/HiSeq Zhong et al, reported 307 haplotypes with 99 predominant and an average MOI of 2.68 from 222 samples collected in Ethiopia, East Africa [24,27].…”