A high-throughput sequencing assay to comprehensively detect and characterize unicellular eukaryotes and helminths from biological and environmental samples

Abstract

BackgroundSeveral of the most devastating human diseases are caused by eukaryotic parasites transmitted by arthropod vectors or through food and water contamination. These pathogens only represent a fraction of all unicellular eukaryotes and helminths that are present in the environment and many uncharacterized organisms might have subtle but pervasive effects on health, including by modifying the microbiome where they reside. Unfortunately, while we have modern molecular tools to characterize bacterial and, to a lesser extent, fungal communities, we lack suitable methods to comprehensively investigate and characterize most unicellular eukaryotes and helminths: the detection of these organisms often relies on microscopy that cannot differentiate related organisms, while molecular assays can only detect the pathogens specifically tested.ResultsHere, we describe a novel sequencing-based assay, akin to bacterial 16S rRNA sequencing, that enables high-throughput detection and characterization of a wide range of unicellular eukaryotes and helminths, including those from taxonomical groups containing all common human parasites. We designed and evaluated taxon-specific PCR primer pairs that selectively amplify all species from eight taxonomical groups (Apicomplexa, Amoeba, Diplomonadida, Kinetoplastida, Parabasalia, Nematoda, Platyhelminthes, and Microsporidia). We then used these primers to screen DNA extracted from clinical, biological, and environmental samples, and after next-generation sequencing, identified both known and previously undescribed organisms from most taxa targeted.ConclusionsThis novel high-throughput assay enables comprehensive detection and identification of eukaryotic parasites and related organisms, from a wide range of complex biological and environmental samples. This approach can be easily deployed to many settings and will efficiently complement existing methods and provide a holistic perspective on the microbiome.Electronic supplementary materialThe online version of this article (10.1186/s40168-018-0581-6) contains supplementary material, which is available to authorized users.

Relative Contributions of Genetic and Non-Genetic Factors on Mosquito Microbiota

“…DNA extracted from each mosquito was ampli ed using primers targeting bacterial 16S rRNA primers [32], mosquito kdr-west (L1014F) [33], cox1 and S200 × 6.1 [34] loci, mammalian mitochondrial 16S rRNA sequences, as well as eukaryotic parasite and virus primers [35] (Additional le 1: Table S1).…”

“…Only 35 cycles were used to amplify bacterial 16S rRNA. All PCR products generated from one DNA sample (i.e., individual mosquito) were pooled together and reampli ed in a second PCR to add a unique barcode and the Illumina sequencing adaptors [35]. Finally, all barcoded products from all mosquitoes were pooled and sequenced simultaneously on Illumina HiSeq 2500 to generate 300 bp paired-end reads [35].…”

“…Then all concatenated sequences ampli ed with the same primer pair (from all samples) were compared to each other and a single copy of each unique sequence was kept (while the number of times it is observed in each sample was recorded). Unique sequences that were observed less than 10 times across all samples were removed as they likely represent instances of sequences carrying errors [35]. The remaining unique sequences were compared against all DNA sequences annotated on the NCBI nr database using BLAST [35].…”

“…Unique sequences that were observed less than 10 times across all samples were removed as they likely represent instances of sequences carrying errors [35]. The remaining unique sequences were compared against all DNA sequences annotated on the NCBI nr database using BLAST [35]. We then retrieved the taxonomic information of the most similar sequence(s) if it had at least 70% identity over the entire sequence length.…”

“…Finally, we determined whether each mosquito carried a eukaryotic parasite and/or arbovirus using a sequencing-based assay recently developed in our laboratory [35]. After PCR ampli cation, and sequencing of DNA extracted from individual mosquitoes, we identi ed DNA sequences from eukaryotes and viruses from 127 mosquitoes, with on average, 1,876 reads supporting each identi cation in each mosquito.…”