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Methods

Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa

Suzanne L. Ishaq, André-Denis G. Wright
J. Björkroth, Editor
Suzanne L. Ishaq
Department of Animal Science, College of Agriculture and Life Sciences, University of Vermont, Burlington, Vermont, USA
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André-Denis G. Wright
Department of Animal Science, College of Agriculture and Life Sciences, University of Vermont, Burlington, Vermont, USA
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J. Björkroth
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DOI: 10.1128/AEM.01644-14
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ABSTRACT

Four new primers and one published primer were used to PCR amplify hypervariable regions within the protozoal 18S rRNA gene to determine which primer pair provided the best identification and statistical analysis. PCR amplicons of 394 to 498 bases were generated from three primer sets, sequenced using Roche 454 pyrosequencing with Titanium, and analyzed using the BLAST database (NCBI) and MOTHUR version 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F and GIC758R (amplicon of 482 bases), gave the best representation of diversity using BLAST classification, and the set amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F and GIC1578R (amplicon of 498 bases), had similar BLAST results and a slightly higher percentage of sequences that were identified with a higher sequence identity. Primer sets 1 and 2 are recommended for use in ruminants. However, primer set 1 may be inadequate to determine protozoal diversity in nonruminants. The amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, and Tetratoxum and between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.

FOOTNOTES

    • Received 17 May 2014.
    • Accepted 23 June 2014.
    • Accepted manuscript posted online 27 June 2014.
  • Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.01644-14.

  • Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa
Suzanne L. Ishaq, André-Denis G. Wright
Applied and Environmental Microbiology Aug 2014, 80 (17) 5515-5521; DOI: 10.1128/AEM.01644-14

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Design and Validation of Four New Primers for Next-Generation Sequencing To Target the 18S rRNA Genes of Gastrointestinal Ciliate Protozoa
Suzanne L. Ishaq, André-Denis G. Wright
Applied and Environmental Microbiology Aug 2014, 80 (17) 5515-5521; DOI: 10.1128/AEM.01644-14
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