A review on application of next-generation sequencing methods for profiling of protozoan parasites in water: Current methodologies, challenges, and perspectives

The advancement in metagenomic techniques has provided novel tools for profiling human parasites in environmental matrices, such as water and wastewater. However, application of metagenomic techniques for the profiling of protozoan parasites in environmental matrices is not commonly reported in the literature. The key factors leading to the less common use of metagenomics are the complexity and large eukaryotic genome, the prevalence of small parasite populations in environmental samples compared to bacteria, difficulties in extracting DNA from (oo)cysts, and limited reference databases for parasites. This calls for further research to develop optimized methods specifically looking at protozoan parasites in the environment. This study reviews the current workflow, methods and provide recommendations for the standardization of techniques. The article identifies and summarizes the key methods, advantages, and limitations associated with metagenomic analysis, like sample pre-processing, DNA extraction, sequencing approaches, and analysis methods. The study enhances the understanding and application of standardized protocols for profiling of protozoan parasite community from highly complexe samples and further creates a resourceful comparison among datasets without any biases. •Sample collection, processing, and DNA extraction steps are critical for metagenomic profiling.•Shotgun and targeted metagenomics sequencing are commonly used for protozoan profiling.•Different variable regions of the 18 s rDNA are targeted for deep amplicon sequencing.•Several pathogenic protozoans have been identified in environmental samples via metagenomics.•Bioinformatics and reference databases are major bottlenecks for metagenome analysis.