{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"medRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T12:20:35Z","timestamp":1768566035904,"version":"3.49.0"},"posted":{"date-parts":[[2023,8,23]]},"group-title":"Infectious Diseases (except HIV\/AIDS)","reference-count":39,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,8,23]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  Molecular epidemiologic studies of malaria parasites commonly employ amplicon deep sequencing (AmpSeq) of marker genes derived from dried blood spots (DBS) to answer public health questions related to topics such as transmission and drug resistance. As these methods are increasingly employed to inform direct public health action, it is important to rigorously evaluate the risk of false positive and false negative haplotypes derived from clinically-relevant sample types. We performed a control experiment evaluating haplotype recovery from AmpSeq of 5 marker genes (\n                  <jats:italic>ama1<\/jats:italic>\n                  ,\n                  <jats:italic>csp<\/jats:italic>\n                  ,\n                  <jats:italic>msp7<\/jats:italic>\n                  ,\n                  <jats:italic>sera2<\/jats:italic>\n                  , and\n                  <jats:italic>trap<\/jats:italic>\n                  ) from DBS containing mixtures of DNA from 1 to 10 known\n                  <jats:italic>P. falciparum<\/jats:italic>\n                  reference strains across 3 parasite densities in triplicate (n=270 samples). While false positive haplotypes were present across all parasite densities and mixtures, we optimized censoring criteria to remove 83% (148\/179) of false positives while removing only 8% (67\/859) of true positives. Post-censoring, the median pairwise Jaccard distance between replicates was 0.83. We failed to recover 35% (477\/1365) of haplotypes expected to be present in the sample. Haplotypes were more likely to be missed in low-density samples with &lt;1.5 genomes\/\u00b5L (OR: 3.88, CI: 1.82-8.27, vs. high-density samples with \u226575 genomes\/\u00b5L) and in samples with lower read depth (OR per 10,000 reads: 0.61, CI: 0.54-0.69). Furthermore, minority haplotypes within a sample were more likely to be missed than dominant haplotypes (OR per 0.01 increase in proportion: 0.96, CI: 0.96-0.97). Finally, in clinical samples the percent concordance across markers for multiplicity of infection ranged from 40%-80%. Taken together, our observations indicate that, with sufficient read depth, haplotypes can be successfully recovered from DBS while limiting the false positive rate.\n                <\/jats:p>","DOI":"10.1101\/2023.08.17.23294237","type":"posted-content","created":{"date-parts":[[2023,8,23]],"date-time":"2023-08-23T14:25:16Z","timestamp":1692800716000},"source":"Crossref","is-referenced-by-count":2,"title":["Analytic optimization of\n                  <i>Plasmodium falciparum<\/i>\n                  marker gene haplotype recovery from amplicon deep sequencing of complex mixtures"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4674-2176","authenticated-orcid":false,"given":"Zena","family":"Lapp","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizabeth","family":"Freedman","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0001-7148-1556","authenticated-orcid":false,"given":"Kathie","family":"Huang","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5742-8847","authenticated-orcid":false,"given":"Christine F","family":"Markwalter","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3730-5484","authenticated-orcid":false,"given":"Andrew A","family":"Obala","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Wendy","family":"Prudhomme-O\u2019Meara","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2783-0990","authenticated-orcid":false,"given":"Steve M","family":"Taylor","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"issue":"1","key":"2023082608050555000_2023.08.17.23294237v1.1","doi-asserted-by":"crossref","first-page":"490","DOI":"10.1186\/s12936-017-2137-9","article-title":"A deep sequencing approach to estimate Plasmodium falciparum complexity of infection (COI) and explore apical membrane antigen 1 diversity","volume":"16","year":"2017","journal-title":"Malaria Journal"},{"key":"2023082608050555000_2023.08.17.23294237v1.2","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-021-21269-2"},{"issue":"8","key":"2023082608050555000_2023.08.17.23294237v1.3","doi-asserted-by":"crossref","first-page":"e0000807","DOI":"10.1371\/journal.pgph.0000807","article-title":"Plasmodium falciparum importation does not sustain malaria transmission in a semi-arid region of Kenya","volume":"2","year":"2022","journal-title":"PLOS Global Public Health"},{"key":"2023082608050555000_2023.08.17.23294237v1.4","doi-asserted-by":"crossref","first-page":"108","DOI":"10.1016\/j.ijid.2023.03.050","article-title":"Evolution of Plasmodium falciparum antimalarial drug resistance markers post-adoption of artemisinin-based combination therapies in Yaounde, Cameroon","volume":"132","year":"2023","journal-title":"International Journal of Infectious Diseases"},{"issue":"4","key":"2023082608050555000_2023.08.17.23294237v1.5","first-page":"e01945","article-title":"Targeted Amplicon Deep Sequencing for Monitoring Antimalarial Resistance Markers in Western Kenya","volume":"66","year":"2022","journal-title":"Antimicrobial Agents and Chemotherapy"},{"issue":"3","key":"2023082608050555000_2023.08.17.23294237v1.6","doi-asserted-by":"crossref","first-page":"788","DOI":"10.1093\/jac\/dkad011","article-title":"Baseline prevalence of molecular marker of sulfadoxine\/pyrimethamine resistance in Ebonyi and Osun states, Nigeria: amplicon deep sequencing of dhps-540","volume":"78","year":"2023","journal-title":"Journal of Antimicrobial Chemotherapy"},{"issue":"1","key":"2023082608050555000_2023.08.17.23294237v1.7","doi-asserted-by":"crossref","first-page":"17790","DOI":"10.1038\/s41598-019-54203-0","article-title":"Amplicon deep sequencing improves Plasmodium falciparum genotyping in clinical trials of antimalarial drugs","volume":"9","year":"2019","journal-title":"Sci Rep"},{"issue":"6","key":"2023082608050555000_2023.08.17.23294237v1.8","first-page":"e01709","article-title":"Amplicon Deep Sequencing Reveals Multiple Genetic Events Lead to Treatment Failure with Atovaquone-Proguanil in Plasmodium falciparum","volume":"67","year":"2023","journal-title":"Antimicrobial Agents and Chemotherapy"},{"key":"2023082608050555000_2023.08.17.23294237v1.9","doi-asserted-by":"publisher","DOI":"10.1186\/s12864-017-4260-y"},{"key":"2023082608050555000_2023.08.17.23294237v1.10","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx1201"},{"issue":"1","key":"2023082608050555000_2023.08.17.23294237v1.11","doi-asserted-by":"crossref","first-page":"219","DOI":"10.1186\/s12936-019-2856-1","article-title":"Detection of low-density Plasmodium falciparum infections using amplicon deep sequencing","volume":"18","year":"2019","journal-title":"Malaria Journal"},{"key":"2023082608050555000_2023.08.17.23294237v1.12","doi-asserted-by":"crossref","unstructured":"LaVerriere E , Schwabl P , Carrasquilla M , Taylor AR , Johnson ZM , Shieh M , et al. Design and implementation of multiplexed amplicon sequencing panels to serve genomic epidemiology of infectious disease: a malaria case study [Internet]. Infectious Diseases (except HIV\/AIDS); 2021 Sep [cited 2021 Sep 27]. Available from: http:\/\/medrxiv.org\/lookup\/doi\/10.1101\/2021.09.15.21263521","DOI":"10.1101\/2021.09.15.21263521"},{"key":"2023082608050555000_2023.08.17.23294237v1.13","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts091"},{"key":"2023082608050555000_2023.08.17.23294237v1.14","doi-asserted-by":"publisher","DOI":"10.4269\/ajtmh.19-0558"},{"issue":"1","key":"2023082608050555000_2023.08.17.23294237v1.15","doi-asserted-by":"crossref","first-page":"5615","DOI":"10.1038\/s41467-019-13578-4","article-title":"High-resolution micro-epidemiology of parasite spatial and temporal dynamics in a high malaria transmission setting in Kenya","volume":"10","year":"2019","journal-title":"Nat Commun"},{"key":"2023082608050555000_2023.08.17.23294237v1.16","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bts480"},{"issue":"1","key":"2023082608050555000_2023.08.17.23294237v1.17","doi-asserted-by":"crossref","first-page":"10","DOI":"10.14806\/ej.17.1.200","article-title":"Cutadapt removes adapter sequences from high-throughput sequencing reads","volume":"17","year":"2011","journal-title":"EMBnet.journal"},{"key":"2023082608050555000_2023.08.17.23294237v1.18","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btu170"},{"key":"2023082608050555000_2023.08.17.23294237v1.19","unstructured":"Bushnell B. BBMap: A Fast, Accurate, Splice-Aware Aligner [Internet]. Lawrence Berkeley National Lab. (LBNL), Berkeley, CA (United States); 2014 Mar [cited 2023 Jul 14]. Report No.: LBNL-7065E. Available from: https:\/\/www.osti.gov\/biblio\/1241166"},{"key":"2023082608050555000_2023.08.17.23294237v1.20","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.3869"},{"key":"2023082608050555000_2023.08.17.23294237v1.21","doi-asserted-by":"publisher","DOI":"10.1186\/1471-2105-12-77"},{"key":"2023082608050555000_2023.08.17.23294237v1.22","first-page":"1","article-title":"Fitting Linear Mixed-Effects Models Using lme4","volume":"67","year":"2015","journal-title":"Journal of Statistical Software"},{"key":"2023082608050555000_2023.08.17.23294237v1.23","unstructured":"R Core Team. R: A Language and Environment for Statistical Computing [Internet]. Vienna, Austria: R Foundation for Statistical Computing; 2021. Available from: https:\/\/www.R-project.org\/"},{"key":"2023082608050555000_2023.08.17.23294237v1.24","unstructured":"RStudio | Open source & professional software for data science teams [Internet]. [cited 2022 Apr 1]. Available from: https:\/\/www.rstudio.com\/"},{"key":"2023082608050555000_2023.08.17.23294237v1.25","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/btv494"},{"issue":"43","key":"2023082608050555000_2023.08.17.23294237v1.26","doi-asserted-by":"crossref","first-page":"1686","DOI":"10.21105\/joss.01686","article-title":"Welcome to the Tidyverse","volume":"4","year":"2019","journal-title":"Journal of Open Source Software"},{"key":"2023082608050555000_2023.08.17.23294237v1.27","unstructured":"Wickham H , Bryan J , attribution) Rs (Copyright holder of all R code and all C code without explicit copyright, code) MK (Author of included R, code) KV (Author of included libxls, code) CL (Author of included libxls, et al. readxl: Read Excel Files [Internet]. 2019 [cited 2022 Feb 16]. Available from: https:\/\/CRAN.R-project.org\/package=readxl"},{"key":"2023082608050555000_2023.08.17.23294237v1.28","doi-asserted-by":"publisher","DOI":"10.1093\/bioinformatics\/bty633"},{"issue":"1","key":"2023082608050555000_2023.08.17.23294237v1.29","first-page":"000747","article-title":". regentrans: a framework and R package for using genomics to study regional pathogen transmission","volume":"8","year":"2022","journal-title":"Microbial Genomics"},{"issue":"1","key":"2023082608050555000_2023.08.17.23294237v1.30","first-page":"1","article-title":"Reshaping Data with the reshape Package","volume":"21","year":"2007","journal-title":"Journal of Statistical Software"},{"key":"2023082608050555000_2023.08.17.23294237v1.31","unstructured":"Wickham H , Seidel D , RStudio. scales: Scale Functions for Visualization [Internet]. 2020 [cited 2022 Apr 1]. Available from: https:\/\/CRAN.R-project.org\/package=scales"},{"key":"2023082608050555000_2023.08.17.23294237v1.32","unstructured":"Wilke CO. cowplot: Streamlined Plot Theme and Plot Annotations for \u201cggplot2\u201d [Internet]. 2019 [cited 2020 Apr 15]. Available from: https:\/\/CRAN.R-project.org\/package=cowplot"},{"key":"2023082608050555000_2023.08.17.23294237v1.33","doi-asserted-by":"crossref","unstructured":"Ahlmann-Eltze C. ggupset: Combination Matrix Axis for \u201cggplot2\u201d to Create \u201cUpSet\u201d Plots [Internet]. 2020 [cited 2023 Jul 14]. Available from: https:\/\/cran.rstudio.com\/web\/packages\/ggupset\/index.html","DOI":"10.32614\/CRAN.package.ggupset"},{"key":"2023082608050555000_2023.08.17.23294237v1.34","doi-asserted-by":"crossref","unstructured":"Bolker B , Robinson D , Menne D , Gabry J , Buerkner P , Hua C , et al. broom.mixed: Tidying Methods for Mixed Models [Internet]. 2019 [cited 2023 Jul 14]. Available from: https:\/\/CRAN.R-project.org\/package=broom.mixed","DOI":"10.32614\/CRAN.package.broom.mixed"},{"key":"2023082608050555000_2023.08.17.23294237v1.35","unstructured":"Aphalo PJ , Slowikowski K. ggpmisc: Miscellaneous Extensions to \u201cggplot2\u201d [Internet]. 2018 [cited 2023 Jul 14]. Available from: https:\/\/CRAN.R-project.org\/package=ggpmisc"},{"key":"2023082608050555000_2023.08.17.23294237v1.36","unstructured":"Kassambara A. ggpubr: \u201cggplot2\u201d Based Publication Ready Plots [Internet]. 2018 [cited 2023 Jul 14]. Available from: https:\/\/CRAN.R-project.org\/package=ggpubr"},{"key":"2023082608050555000_2023.08.17.23294237v1.37","doi-asserted-by":"crossref","unstructured":"Wilke CO. ggtext: Improved Text Rendering Support for \u201cggplot2\u201d [Internet]. 2020 [cited 2022 Apr 1]. Available from: https:\/\/CRAN.R-project.org\/package=ggtext","DOI":"10.32614\/CRAN.package.ggtext"},{"key":"2023082608050555000_2023.08.17.23294237v1.38","unstructured":"An open dataset of Plasmodium falciparum \u2026 | Wellcome Open Research [Internet]. [cited 2023 Jul 14]. Available from: https:\/\/wellcomeopenresearch.org\/articles\/6-42"},{"key":"2023082608050555000_2023.08.17.23294237v1.39","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkv717"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2023.08.17.23294237","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T19:39:17Z","timestamp":1768505957000},"score":1,"resource":{"primary":{"URL":"http:\/\/medrxiv.org\/lookup\/doi\/10.1101\/2023.08.17.23294237"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,23]]},"references-count":39,"URL":"https:\/\/doi.org\/10.1101\/2023.08.17.23294237","relation":{},"subject":[],"published":{"date-parts":[[2023,8,23]]},"subtype":"preprint"}}