An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads

Abstract

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.Electronic supplementary materialThe online version of this article (doi:10.1007/s00248-017-0996-9) contains supplementary material, which is available to authorized users.

Comparative analyses of the V4 and V9 regions of 18S rDNA for the extant eukaryotic community using the Illumina platform

“…Although the average values are not significantly different from each other (t-test, p = 0.752), the average Shannon diversity and inverse Simpson indexes for V4 region is slightly higher than those for the V9 region. Several previous studies reported that the Shannon diversity and inverse Simpson indexes for V4 region were lower than those for V9 region 20,21,33 . Conversely, Hirakata et al 30 reported that the Shannon diversity and inverse Simpson indexes for V4 region were higher than those for the V9 region, consistent with our results.…”

“…The determination of the identity threshold is one of the critical steps affecting the number of examined OTUs 21,23 . At the higher identity threshold, the estimation of diversity in eukaryotes is more conservative 13,20 . Surprisingly, Tragin et al 21 and Piredda et al 8 demonstrated that the fraction of the Figure 5.…”

“…Atlantic Ocean, Pacific Ocean, and the Mediterranean Sea). Additionally, Maritz et al 20 reported that the V9 region OTUs (i.e. 1,444 OTUs) were 27% higher than the V4 region OTUs (i.e.…”

Overlapping Community Compositions of Gut and Fecal Microbiomes in Lab-Reared and Field-Collected German Cockroaches

“…Demultiplexed reads were subjected to de novo chimera checking and removal of singletons, and they were clustered into OTUs at 98% identity following the UPARSE pipeline (USEARCH version 8.0.1) ( 86 ). Taxonomy was assigned to representative OTU sequences using BLAST ( 87 ) within QIIME, first against a curated SILVA database ( 88 ) and subsequently with the QIIME-formatted SILVA 111 database clustered at 99% identity ( 89 ). OTUs with no significant hits (<90% identity) after both rounds of taxonomic assignment were labeled as “unidentified.” OTUs were then filtered to remove non-18S rRNA gene sequences (bacterial and archaeal OTUs), host sequences ( B. germanica ), and low-abundance OTUs making up <0.001% of reads in the total data set, as recommended for Illumina sequencing data ( 90 ).…”

Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution

“…Operational taxonomic unit (OTU) clustering was carried out in this study using a 98% clustering identity threshold that is more stringent than the more commonly used 97% threshold. OTU clustering at 97% has recently been shown to be too conservative for estimating diversity in microbial eukaryotes, merging different species in the same cluster [ 56 , 57 ]. Further, a 97% clustering identity is usually applied to illumina amplicons when targeting variable rRNA regions [ 58 ], with a more stringent threshold being traditionally applied to longer read rRNA amplicons encompassing conserved and variable regions [ 56 , 59 ].…”