Abstract
We recently described a targeted amplicon deep sequencing (TADS) strategy that utilizes a nested PCR targeting the 18S rDNA gene of blood-borne parasites. The assay facilitates selective digestion of host DNA by targeting enzyme restriction sites present in vertebrates but absent in parasites. This enriching of parasite-derived amplicon drastically reduces the proportion of host-derived reads during sequencing and results in the sensitive detection of several clinically important blood parasites including Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes. Despite these promising results, high costs and the laborious nature of metagenomics sequencing are prohibitive to the routine use of this assay in most laboratories. We describe and evaluate a new metagenomic approach that utilizes a set of primers modified from our original assay that incorporates Illumina barcodes and adapters during the PCR steps. This modification makes amplicons immediately compatible with sequencing on the Illumina MiSeq platform, removing the need for a separate library preparation, which is expensive and time-consuming. We compared this modified assay to our previous nested TADS assay in terms of preparation speed, limit of detection (LOD), and cost. Our modifications reduced assay turnaround times from 7 to 5 days. The cost decreased from approximately $40 per sample to $11 per sample. The modified assay displayed comparable performance in the detection and differentiation of human-infecting Plasmodium spp., Babesia spp., kinetoplastids, and filarial nematodes in clinical samples. The LOD of this modified approach was determined for malaria parasites and remained similar to that previously reported for our earlier assay (0.58 Plasmodium falciparum parasites/µL of blood). These modifications markedly reduced costs and turnaround times, making the assay more amenable to routine diagnostic applications.
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Data availability
Raw reads analyzed in this study have been made publicly available by submission to NCBI Sequence Read Archive and can be accessed under BioProject accession number: PRJNA437674. BioSamples submitted to this BioProject that are relevant to the present study include the term “Ad_UPDx vs nUPDx” in their sample name..
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Acknowledgements
The authors thank Brianna Flaherty, former ORISE fellow at CDC, Stella Chenet and Maria Isabel Jercic from the Institute of Public Health, Chile, and Andrew Moorhead from the University of Georgia for providing samples. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.
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This study was made possible by support from the Centers for Disease Control and Prevention Advanced Molecular Detection (AMD) Initiative.
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MG optimized the experimental design, performed test experiments, analyzed the data, generated the figures, and drafted the manuscript. ML performed LOD, DNA library preparations, and Illumina sequencing experiments. JB assisted with the conception of the method, data analysis, and editing of the manuscript. ET assisted with the conception of the method and experimental design. YQ supervised the study and edited the manuscript. All authors read and approved the final manuscript.
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Ethics approval for the use of anonymized or de-identified blood samples as exempt human research was granted by the Centers for Disease Control and Prevention Human Research Protection Office (protocol #6756).
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Gondard, M., Lane, M., Barratt, J. et al. Simultaneous targeted amplicon deep sequencing and library preparation for a time and cost-effective universal parasite diagnostic sequencing approach. Parasitol Res 122, 3243–3256 (2023). https://doi.org/10.1007/s00436-023-07991-4
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DOI: https://doi.org/10.1007/s00436-023-07991-4