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    Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci

    Access Status
    Fulltext not available
    Authors
    Paparini, A.
    Gofton, A.
    Yang, R.
    White, Nicole
    Bunce, Michael
    Ryan, U.
    Date
    2015
    Type
    Journal Article
    
    Metadata
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    Citation
    Paparini, A. and Gofton, A. and Yang, R. and White, N. and Bunce, M. and Ryan, U. 2015. Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci. Experimental Parasitology. 151-152: pp. 21-27.
    Source Title
    Experimental Parasitology
    DOI
    10.1016/j.exppara.2015.02.001
    ISSN
    00144894
    School
    Department of Environment and Agriculture
    URI
    http://hdl.handle.net/20.500.11937/7959
    Collection
    • Curtin Research Publications
    Abstract

    Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrentbasedgenotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing.

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