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

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. Rumen ciliate protozoa represent important functional members of the rumen environment, as most have some cellulolytic or amylolytic abilities (1-3). Most studies of rumen ciliate protozoa are performed using microscopy and traditional culturing techniques (2, 4-10), quantitative PCR (11, 12), denaturing gradient gel electrophoresis (13), and full-length 18S rRNA clone libraries (13,14). A few studies of rumen ciliate protozoa use highthroughput sequencing, although primer selection remains a problem, as some studies use universal eukaryotic primers, primers which target only one ciliate protozoon signature region, or primers which produce long amplicons that are unsuitable for current high-throughput technology (15)(16)(17)(18)(19)(20).18S rRNA genes range from 1.5 kb to more than 4.5 kb (21), and in rumen ciliate protozoa, they are generally 1.5 kb to 1.8 kb in length. Like the 16S rRNA genes of prokaryotes, the 18S rRNA genes of eukaryotes have nine hypervariable regions (V1 to V9) which can be used for genus/species identification. Four gut ciliate signature regions exist within rumen protozoal 18S rRNA genes which represent areas of high variability that can improve identification down to the species level (22, 23). Signature region 1 occurs between bp 440 and 460 (within V3), signature region 2 occurs between bp 590 and 620 (between V3 and V4), signature region 3 occurs between bp 1220 and 1260 (within V6), and signature region 4 occurs between bp 1560 and 1580 (after V8) (Fig. 1). Additionally, rumen ciliate protozoa have a slightly different 18S rRNA secondary structure from nonrumen ciliates, in that rumen protozoa are missing helix E23-5 from the V4 region and other hel...

Genetic Analysis of Babesia Isolates from Cattle with Clinical Babesiosis in Sri Lanka

“…Single forms were often elongated or irregularly shaped. 18S rRNA is the most commonly used molecular marker for species identification of eukaryotes, including Babesia (18,20,(45)(46)(47). Therefore, in an attempt to identify the species of Babesia detected in cow R, a 1,385-bp 18S rRNA sequence was isolated from cow R (GenBank accession number LC385886) and compared with the Babesia sequences in the GenBank database.…”

Phylogeny of Intestinal Ciliates, Including Charonina ventriculi, and Comparison of Microscopy and 18S rRNA Gene Pyrosequencing for Rumen Ciliate Community Structure Analysis

“…(27) and overestimates of the abundance of, e.g., Polyplastron spp. (74). For this reason, the pyrosequencing method cannot be used to estimate ␣-diversity or relative abundances of different genera and species in a sample.…”

Extending Burk Dehority’s Perspectives on the Role of Ciliate Protozoa in the Rumen

“…This potential limitation certainly applies to PCR amplicon-based analysis of rumen protozoa. Among the protozoa-specific primers targeting the 18S rRNA genes, most studies used primers RP841F (5 -GACTAGGGATTGGAGTGG-3 ) and Reg1302R (5 -AATTGCAAAGATCTATCCC-3 ) targeting the V5-V8 regions (Regensbogenova et al, 2004;Kittelmann and Janssen, 2011) and P-SSU-316F (5 -GCTTTCGWTGGTAGTGTATT-3 ) and GIC758R (5 -CAACTGTCTCTATKAAYCG-3 ) covering two signature regions of rumen ciliates (Sylvester et al, 2004;Ishaq and Wright, 2014). The RP841F/Reg1302R primer set was developed initially for DGGE analysis and designed from only the 18S rDNA sequence of rumen ciliates (Kittelmann and Janssen, 2011).…”

“…The P-SSU-316F/GIC758R primer set was designed for amplicon sequencing using NGS. It was evaluated for its specificity for rumen protozoa against protozoal reference 18S rRNA gene sequences, including those of non-ruminal ciliates that were available in NCBI (Ishaq and Wright, 2014). Both primer sets allowed detection and identification of the major ruminal ciliates at the genus taxonomic rank and detected similar diversity as microscopic identification; however, rather different protozoal relative abundance resulted from the two primer sets.…”

“…In general, small entodiniomorphids have fewer 18S rRNA gene copies per cell than larger cells (Sylvester et al, 2009). Therefore, the absolute abundance (copies of 18S rRNA genes per unit of weight or volume of samples) and relative abundance (% of total protozoal sequences) of small entodinia can be underestimated by qPCR and amplicon sequencing, respectively, whereas those with a large cell, such as Polyplastron and Epidinium, are overestimated (Ishaq and Wright, 2014;Kittelmann et al, 2015). As such, 18S rRNA gene-targeting qPCR probably does not allow accurate quantification of protozoal abundance per se but might be associated more positively with activity if we can assume that larger cells have greater activity on a cell basis (Wenner et al, 2018).…”