Untargeted metagenomics shows a reliable performance for synchronous detection of parasites

Abstract

Shotgun metagenomics with high-throughput sequencing (HTS) techniques is increasingly used for pathogen identification and characterization. While many studies apply targeted amplicon sequencing, here we used untargeted metagenomics to simultaneously identify protists and helminths in pre-diagnosed faecal and tissue samples. The approach starts from RNA and operates without an amplification step, therefore allowing the detection of all eukaryotes, including pathogens, since it circumvents the bias typically observed in amplicon-based HTS approaches. The generated metagenomics datasets were analysed using the RIEMS tool for initial taxonomic read assignment. Mapping analyses against ribosomal reference sequences were subsequently applied to extract 18S rRNA sequences abundantly present in the sequence datasets. The original diagnosis, which was based on microscopy and/or PCR, could be confirmed in nearly all cases using ribosomal RNA metagenomics. In addition to the prediagnosed taxa, we detected other intestinal eukaryotic parasites of uncertain pathogenicity (of the genera Dientamoeba, Entamoeba, Endolimax, Hymenolepis) that are often excluded from routine diagnostic protocols. The study clearly demonstrates the applicability of untargeted RNA metagenomics for the parallel detection of parasites.

Parasitic Intestinal Protists of Zoonotic Relevance Detected in Pigs by Metabarcoding and Real-Time PCR

“…Interestingly, Giardia was not identified in any of the pig samples subject to shotgun sequencing by Wylezich et al [16]. That team recently published an evaluation of their method [38] and mentioned the low recovery rate for Giardia as a limitation, which means that Giardia may have been overlooked in their study of pigs [16], and thus, it is worth noticing that Giardia may generally be difficult to detect not only by the metabarcoding methods but also by a metagenomics approach. To this end, Ramayo-Caldas et al [17] used amplicon-based NGS relying on detection and differentiation of ITS and 18S rRNA fragments and mentioned neither Giardia nor Cryptosporidium among their findings.…”

Meta-Ribosomalomics: RNA Sequencing Is an Unbiased Method for Parasite Detection of Different Sample Types

“…Another advantage (see Table 1 ) is the complete absence of any primer bias since no target specific primers are applied to amplify target genes. Untargeted (PCR- and primer-free) approaches are well suited to catch hitherto unknown, unrelated or unexpected sequence types ( Wylezich et al, 2019a , b , 2020 ).…”

“…In the above-mentioned examples, the generic meta-ribosomalomics approach was promising for diagnostics of typical but also neglected parasites and gut protists (e.g., Wylezich et al, 2019a , b , 2020 ). In addition, ribosomal gene sequences can be retrieved from DNA-based metagenomics datasets as shown for a novel species of ammonia-oxidizing Archaea ( Zou et al, 2020 ).…”

“… Milestones of generating small-subunit ribosomal RNA gene sequences of prokaryotes and protistan eukaryotes to address phylogenetic and ecological questions (with references for the first application each; Fox and Woese, 1975 ; Brosius et al, 1978 ; McCarroll et al, 1983 ; Spangler and Blackburn, 1985 ; Medlin et al, 1988 ; Edwards et al, 1989 ; Giovannoni et al, 1990 ; Moon-van der Staay et al, 2001 ; Sogin et al, 2006 ; Amaral-Zettler et al, 2009 ; Wylezich et al, 2019a , b , 2020 ). …”

“…Hence, a hypothesis-free approach can be important to uncover the full diversity of species present in a sample which could be overlooked in case an expected species was detected and the analysis for other species stopped thereafter. In a follow-up study, the method was applied to fecal and organ samples pre-diagnosed for parasites ( Wylezich et al, 2020 ). Diagnoses could be confirmed in nearly all cases.…”