With the advent of low-cost, high-throughput sequencing, taxonomic profiling of complex microbial communities through 16S rRNA marker gene surveys has received widespread interest, uncovering a wealth of information concerning the bacterial composition of microbial communities, as well as their association with health and disease. On the other hand, little is known concerning the eukaryotic components of microbiomes. Such components include single-celled parasites and multicellular worms that are known to adversely impact the health of millions of people worldwide. Current molecular methods to detect eukaryotic microbes rely on the use of directed PCR analyses that are limited by their inability to inform beyond the taxon targeted. With increasing interest to develop equivalent marker-based surveys as used for bacteria, this chapter presents a stepwise protocol to characterize the diversity of eukaryotic microbes in a sample, using amplicon sequencing of hypervariable regions in the eukaryotic 18S rRNA gene.
Keywords: 18S rRNA; Amplicon sequencing; Eukaryotic microbes; Illumina MiSeq; Microbiome; Taxonomic assignment.