Sensitive, highly multiplexed sequencing of microhaplotypes from thePlasmodium falciparumheterozygome

Abstract

The first two authors contributed equally to this work. ✝ The last two authors contributed equally to this work. AbstractBackground: Targeted next generation sequencing offers the potential for consistent, deep coverage of information rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. Methods:A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, WGS data were generated for the same controls.Results: Using publicly available genomes, 4465 high diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median HE = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%) and sensitivity (90% for haplotypes with 5% within sample frequency in DBS with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic SNP barcodes. Discussion:The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

Highly sensitive and full-genome interrogation of SARS-CoV-2 using multiplexed PCR enrichment followed by next-generation sequencing

“…Multiplex PCR has been shown to be an efficient and low-cost method to detect Plasmodium falciparum infections, with high coverage (median 99%), specificity (99.8%) and sensitivity. Moreover, this solution can be tailored to simultaneously address multiple questions of interest within various epidemiological settings 42 . Similar to a recently described metagenomic approach for SARS-CoV-2 identification 43 , we also establish a user-friendly multiplex-PCR-based metagenomic method that is not only able to detect SARS-CoV-2, but could also be applied for the identification of significant sequence mutations within known viruses and to uncover novel pathogens with a limited sequencing depth of approximately 1 million reads.…”

Within-Household Clustering of Genetically Related Plasmodium Falciparum Infections In A Moderate Transmission Area of Uganda

“…Other genotyping techniques that are able to resolve multiallelic loci, such as multiplex amplicon or "microhaplotype" sequencing, can provide rich data from polyclonal infections and leverage current sequencing technologies to more easily allow evaluation of larger numbers of loci, providing higher resolution to evaluate transmission particularly in moderate-high transmission settings. (25,(31)(32)(33)(34) While these methods are more expensive and require more extensive laboratory and bioinformatics structure than the methods used in this study, they may be preferred techniques in the future as sequencing costs continue to decline.…”

Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: a malaria case study

“…We compared AMPLseq and 4CAST to two previously published AmpSeq panels for malaria molecular surveillance, Paragon HeOME v1 (Tessema et al, 2020) and SpotMalaria v2 (Jacob et al, 2021) .…”

“…We used the R package paneljudge to assess in silico the impact of choosing a specific genotyping panel for relatedness inference. Considering the choice of panel, we evaluated relatedness estimation from data simulated on our 4CAST and AMPLseq panels, the SpotMalaria v2 (Jacob et al, 2021) and Paragon HeOME v1 (Tessema et al, 2020) amplicon panels, and a barcode of 24 SNPs. When data were simulated using microhaplotype frequency estimates of Senegalese parasites, we found that almost all estimates of unrelated or clonal pairs were correctly classified, regardless of the panel ( Fig.…”

“…However, the cost and technical challenges of generating, storing, and interpreting WGS data are impediments to scalability and widespread implementation for organisms with large genomes, or microbes with small genomes in samples dominated by host DNA. Targeted sequencing approaches that focus deep coverage on select genomic regions of interest using multiplexed PCR amplification (AmpSeq) are finding increased application in conservation genomics and fisheries biology (Baetscher, Clemento, Ng, Anderson, & Garza, 2018;Hargrove, McCane, Roth, High, & Campbell, 2021;Natesh et al, 2019;Schmidt, Campbell, Govindarajulu, Larsen, & Russello, 2020) , and can serve genomic epidemiology of infectious diseases by focusing sequencing coverage on the most informative regions of pathogen or parasite genomes, instead of typically dominant host genomes (Jacob et al, 2021;Tessema et al, 2020) .…”