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

Abstract

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.Results 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.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.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4260-y) contains supplementary material, which is available to authorized users.

Malaria Epidemiology at the Clone Level

“…This can be overcome by deep-sequencing highly diverse amplicons containing several SNPs. Diversity of such amplicons is comparable to microsatellites [18, 89, 90], and for P. falciparum , sensitivity to detect minority clones was greatly improved compared to size polymorphic markers [90]. However, amplicon sequencing raw data analysis is more laborious than microsatellite analysis; particularly the distinction between sequencing errors and true minority clones can be challenging.…”

“…Amplicon sequencing is the only technology that can identify previously unknown SNPs, which is of particular relevance in the light of the very high diversity of malaria parasites. It is also the only method that may allow reliable reconstruction of several multi-locus haplotypes in multiclone infections, as read numbers represent the proportion of each clone in a mixed infection with high accuracy [89, 90]. This is difficult with microsatellite data; the minority clone might not be detected due to template competition in the PCR, and due to preferred amplification of smaller alleles, peak height does not always reflect template density [35].…”

Sensitive, highly multiplexed sequencing of microhaplotypes from thePlasmodium falciparumheterozygome

“…Recent advances in next-generation sequencing (NGS) allow targeted deep sequencing of short, highly variable regions with numerous alleles (microhaplotypes) [16,17], predominantly composed of 3 or more SNPs, allowing for detailed characterization of the ensemble of parasites in an infection. Most applications of these methods to date have targeted one or a few genomic loci to provide information on drug resistance, composition of infections or selection [18][19][20][21][22][23][24][25]. Extending these methods to numerous genetically diverse loci offers the potential for high resolution comparisons of infections at a population level [17].…”

Detection of low-density Plasmodium falciparum infections using amplicon deep sequencing

“…Correctly identifying sequence errors is therefore a challenge when applying amplicon sequencing to P. falciparum. Fortunately, several new analytical tools have been developed in recent years to address these challenges [18][19][20][21]. Unlike approaches that use reference datasets or cluster sequences with hard percent-identity thresholds, these new methods are more flexible and can distinguish among sequences that differ by only a single nucleotide change [22].…”

“…This study assesses amplicon sequencing's lower limit of detection using four analysis tools, and further evaluates each tool's accuracy and capacity to recover quantitative information on the relative abundance of different haplotypes within infections. Three of these tools-DADA2 [18], HaplotypR [19], and SeekDeep [20]-were previously published and developed to contend with any Plasmodium amplicon. The fourth-the Parallel Amplicon Sequencing Error Correction (PASEC) pipeline-is a distance-and abundance-based error-correction tool that was specifically tailored for use with CSP and SERA2 amplicons [1] and is formally presented here for the first time.…”