• Login
    View Item 
    •   UMB Digital Archive
    • UMB Open Access Articles
    • UMB Open Access Articles
    • View Item
    •   UMB Digital Archive
    • UMB Open Access Articles
    • UMB Open Access Articles
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of UMB Digital ArchiveCommunitiesPublication DateAuthorsTitlesSubjectsThis CollectionPublication DateAuthorsTitlesSubjects

    My Account

    LoginRegister

    Statistics

    Display statistics

    Optimization of parasite DNA enrichment approaches to generate whole genome sequencing data for Plasmodium falciparum from low parasitaemia samples

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Author
    Shah, Z.
    Adams, M.
    Moser, K.A.
    Shrestha, B.
    Stucke, E.M.
    Laufer, M.K.
    Serre, D.
    Silva, J.C.
    Takala-Harrison, S.
    Date
    2020
    Journal
    Malaria journal
    Publisher
    Springer Nature
    Type
    Article
    
    Metadata
    Show full item record
    See at
    https://doi.org/10.1186/s12936-020-03195-8
    Abstract
    BACKGROUND: Owing to the large amount of host DNA in clinical samples, generation of high-quality Plasmodium falciparum whole genome sequencing (WGS) data requires enrichment for parasite DNA. Enrichment is often achieved by leukocyte depletion of infected blood prior to storage. However, leukocyte depletion is difficult in low-resource settings and limits analysis to prospectively-collected samples. As a result, approaches such as selective whole genome amplification (sWGA) are being used to enrich for parasite DNA. However, sWGA has had limited success in generating reliable sequencing data from low parasitaemia samples. In this study, enzymatic digestion with MspJI prior to sWGA and whole genome sequencing was evaluated to determine whether this approach improved genome coverage compared to sWGA alone. The potential of sWGA to cause amplification bias in polyclonal infections was also examined. METHODS: DNA extracted from laboratory-created dried blood spots was treated with a modification-dependent restriction endonuclease, MspJI, and filtered via vacuum filtration. Samples were then selectively amplified using a previously reported sWGA protocol and subjected to WGS. Genome coverage statistics were compared between the optimized sWGA approach and the previously reported sWGA approach performed in parallel. Differential amplification by sWGA was assessed by comparing WGS data generated from lab-created mixtures of parasite isolates, from the same geographical region, generated with or without sWGA. RESULTS: MspJI digestion did not enrich for parasite DNA. Samples that underwent vacuum filtration (without MspJI digestion) prior to sWGA had the highest parasite DNA concentration and displayed greater genome coverage compared to MspJI?+?sWGA and sWGA alone, particularly for low parasitaemia samples. The optimized sWGA (filtration?+?sWGA) approach was successfully used to generate WGS data from 218 non-leukocyte depleted field samples from Malawi. Sequences from lab-created mixtures of parasites did not show evidence of differential amplification of parasite strains compared to directly sequenced samples. CONCLUSION: This optimized sWGA approach is a reliable method to obtain WGS data from non-leukocyte depleted, low parasitaemia samples. The absence of amplification bias in data generated from mixtures of isolates from the same geographic region suggests that this approach can be appropriately used for molecular epidemiological studies.
    Keyword
    Malaria
    Plasmodium falciparum
    Selective whole genome amplification
    Vacuum filtration
    Whole genome sequencing
    Identifier to cite or link to this item
    https://www.scopus.com/inward/record.uri?eid=2-s2.0-85082790753&doi=10.1186%2fs12936-020-03195-8&partnerID=40&md5=b42acae35263869e71d75dbbf663d053; http://hdl.handle.net/10713/12543
    ae974a485f413a2113503eed53cd6c53
    10.1186/s12936-020-03195-8
    Scopus Count
    Collections
    UMB Open Access Articles

    entitlement

     
    DSpace software (copyright © 2002 - 2022)  DuraSpace
    Quick Guide | Policies | Contact Us | UMB Health Sciences & Human Services Library
    Open Repository is a service operated by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.