Evaluating the potential of direct RNA nanopore sequencing: Metatranscriptomics highlights possible seasonal differences in a marine pelagic crustacean zooplankton community

Abstract

The implementation of cost-effective monitoring programs for zooplankton remains challenging due to the requirements of taxonomical expertise and the high costs of sampling and species identification. To reduce costs, molecular methods have been proposed as alternatives to morphology-based monitoring. Metatranscriptomics can contribute to promote both cost-effectiveness and accuracy of biological assessments of aquatic ecosystems. Here, we describe and evaluate the construction of a metatranscriptome dataset from a pelagic crustacean zooplankton community. We sampled zooplankton in one marine station, named LW02, in the North Sea, in both winter and summer, and generated transcripts using Oxford Nanopore Technology (ONT), a third-generation nanopore-based sequencing technology. ONT is, uniquely, capable of sequencing RNA directly, rather than depending on reverse transcription and PCR, and applicable to be used directly in the field. We found that metatranscriptomics is capable of species detection, including screening for the presence of endoparasites, hence competing with morphological identification. Taxonomic analysis based on ribosomal 18S transcripts identified calanoid copepods, particularly Temora longicornis and Acartia clausi, as the most abundant community members. Moreover, up to 40.4% and 50.5% of all sequences could be assigned to predicted genes in the winter and summer sample, respectively. The most abundant mRNA transcripts with known function coded for essential metabolic processes. GO term annotation revealed that genes involved in glycolytic and translation-related processes were most expressed in the community. Although small in scale, our study provides the basis for future efforts to characterize the metatranscriptome of marine zooplankton communities and its application in biomonitoring programs.

Nanopore sequencing and its application to the study of microbial communities

“…Whereas some well-known pipelines for short-read metagenomics, such as MEGAN [30] and MG-RAST [109] , can be used with RNA reads for taxonomic assignment, the availability of specific tools to analyze long-read RNA profiles is limited. Metataxonomic workflows, as described elsewhere [110] , can be performed alternatively by extracting rRNA sequences, such as the small subunits (16S/18S) and large subunits (23S/28S), using specialized software, e.g., METAXA2 [111] . Functional analysis is performed with BLASTx or Magic-BLAST [112] to align the RNA sequences to a protein database in order to assign either Gene Ontology terms (GO) with Blast2GO [113] or metabolic pathway annotations according to KEGG [114] .…”

Marine Microeukaryote Metatranscriptomics: Sample Processing and Bioinformatic Workflow Recommendations for Ecological Applications

“…In contrast to the Illumina library preparation process in which RNA is required to be converted to cDNA, third generation sequencing technology can sequence RNA molecules directly. These applications to the marine environment are nascent and still being developed, with current limitations including relatively high read error rate and inter-run variability (Semmouri et al, 2020). However, preliminary findings show successful application to marine pelagic zooplankton communities, with high predicted protein content (Semmouri et al, 2020).…”

Direct RNA Nanopore Sequencing of Pseudomonas aeruginosa Clone C Transcriptomes

“…Despite these obvious advantages, the amount of direct RNA-seq data among published transcriptome studies is small, which could partly be attributed to the lack of convenient software pipelines for the processing and evaluation of the primary sequencing data ( 24 ). Within the microbial world, the main subjects of the few publications have been lower eukaryotes or RNA viruses ( 25 – 28 ). We identified only one bacteriological study that applied direct RNA-seq to determine the expression of the resistome in extensively drug-resistant Klebsiella pneumoniae isolates ( 29 ).…”