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Background: Diverse communities of microbial eukaryotes in the global ocean provide a variety of essential ecosystem services, from primary production and carbon flow through trophic transfer to cooperation via symbioses. Increasingly, these communities are being understood through the lens of omics tools, which enable high-throughput processing of diverse communities. Metatranscriptomics offers an understanding of near real-time gene expression in microbial eukaryotic communities, providing a window into community metabolic activity. Results: Here we present a workflow for eukaryotic metatranscriptome assembly, and validate the ability of the pipeline to recapitulate real and manufactured eukaryotic community-level expression data. We also include an open-source tool for simulating environmental metatranscriptomes for testing and validation purposes. We reanalyze previously published metatranscriptomic datasets using our metatranscriptome analysis approach. Conclusion: We determined that a multi-assembler approach improves eukaryotic metatranscriptome assembly based on recapitulated taxonomic and functional annotations from an in-silico mock community. The systematic validation of metatranscriptome assembly and annotation methods provided here is a necessary step to assess the fidelity of our community composition measurements and functional content assignments from eukaryotic metatranscriptomes. [ABSTRACT FROM AUTHOR]

The x-axis indicates the annotations assigned to each of the coding sequences, and the y-axis shows the between-sample sum of reads assigned to coding sequences for that category. These are from the "Not in MATOU" "ft" and "t" bars in Panel E. Dinoflagellated dominate many of the Southern Ocean samples, particularly for those coding sequences which could not be taxonomically annotated The original article [[1]] has been corrected. The y-axis shows the color-coded Tara Oceans sample. [Extracted from the article]