Design and validation of four new primers for next-generation sequencing to target the 18S rRNA gene of gastrointestinal ciliate protozoa.

If the research tools you require don’t exist- then you must create them yourself.  Such is often the case in working with microbial genomics.  In order to adapt sequencing technology to identify rumen ciliate protozoa, we needed to first design primers which to be used for Polymerase Chain Reaction (PCR) in order to amplify enough copies of the 18S rRNA gene for laboratory work.  This involved designing primers in silico, by aligning sequences from the few protozoal 18S rRNA genes publicly-available at the time, in order to identify short sections which were identical across protozoal species.  We then added 18S rRNA gene sequences to our alignment from other Eukaryotes, such as fungi and plants, which we did not want to amplify, to ensure that our primers would target only the desired taxa.  We also needed to design a primer set which would work well in the laboratory; in particular which had an optimal size for the sequencing technology on hand, and which would provide enough information in the portion of the gene capture to identify which protozoal species the DNA in our rumen samples originated from.

Ishaq, S.L., Wright, A-D.G. 2014. Design and validation of four new primers for next-generation sequencing to target the 18S rRNA gene of gastrointestinal ciliate protozoa. Applied and Environmental Microbiology, 80(17):5515-5521.  Article

Four new primers and one published primer were used to PCR amplify hyper-variable 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 (NCBI) database and MOTHUR ver. 1.29. The protozoal diversity of rumen contents from moose in Alaska was assessed. In the present study, primer set 1, P-SSU-316F + GIC758R (amplicon = 482 bases) gave the best representation of diversity using BLAST classification, and amplified Entodinium simplex and Ostracodinium spp., which were not amplified by the other two primer sets. Primer set 2, GIC1080F + GIC1578R (amplicon = 498 bases), had similar BLAST results and a slightly higher percentage of sequences that 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 non-ruminants. Amplicons created by primer set 1 were indistinguishable for certain species within the genera Bandia, Blepharocorys, Polycosta, Tetratoxum, or between Hemiprorodon gymnoprosthium and Prorodonopsis coli, none of which are normally found in the rumen.


Figure 1: A map of the full-length protozoal 18S rRNA gene, including variable (V1-V9) and rumen ciliate signature regions (SR1-SR4), and showing the respective amplicons of the three primer sets used in the present study.

Figure 2: Taxonomy and proportion of unique pyrosequences using NCBI (BLAST), by forward primers P-SSU-316F (Sylvester et al., 2004), GIC1080F (present study), and GIC1184F (present study). All sequences used passed all quality assurance steps outlined in Methods.

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