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

Abstract

Microeukaryotes (protists) serve fundamental roles in the marine environment as contributors to biogeochemical nutrient cycling and ecosystem function. Their activities can be inferred through metatranscriptomic investigations, which provide a detailed view into cellular processes, chemical-biological interactions in the environment, and ecological relationships among taxonomic groups. Established workflows have been individually put forth describing biomass collection at sea, laboratory RNA extraction protocols, and bioinformatic processing and computational approaches. Here, we present a compilation of current practices and lessons learned in carrying out metatranscriptomics of marine pelagic protistan communities, highlighting effective strategies and tools used by practitioners over the past decade. We anticipate that these guidelines will serve as a roadmap for new marine scientists beginning in the realms of molecular biology and/or bioinformatics, and will equip readers with foundational principles needed to delve into protistan metatranscriptomics.

Seasonal Metabolic Dynamics of Microeukaryotic Plankton: A Year-long Metatranscriptomic Study in a Temperate Sea

“…Relative abundance of a given taxonomic group was calculated as the sum of all TPM values belonging to that level divided by the total TPM sum of all taxonomically annotated transcripts for that sample or month, ignoring unannotated transcripts. Transcripts per L were calculated by relating the TPM counts to the spike-in recovery values while accounting for sample processing volumes 26 (see Supplementary Methods).…”

“…Where Copies i,j L -1 equals the amount of RNA copies per L of seawater of transcript i in sample j , reads i,j are the TPM values of read i in sample j , spike copies j is the average amount of copies added to sample j , spike reads j are the average amount of spike reads (TPM) in sample j , volume filtered j is the volume of seawater filtered for sample j , and resuspension volume j is the amount of seawater used to re-elute the filtration residue 4 . In calculating the amount of RNA copies per L of seawater we ignored the loss of extracted RNA for quality control, as these volumes were equal for all samples.…”

“…However, many studies only track natural microeukaryotic plankton assemblages over limited periods of time 21 , or only at a few dates of the seasonal cycle 22 , whereas complete seasonal trajectories are needed to fully understand the temporal dynamics of plankton assemblages and the associated metabolic functions. Metatranscriptomic profiling provides an underused opportunity for a more comprehensive characterization of coastal microeukaryote plankton ecosystems [23][24][25][26] . The systematic capture of microeukaryotic plankton gene expression profiles over time from fixed locations generates ecosystem snapshots that facilitate the reconstruction of the turnover and relationships of active species and their metabolic responses to environmental fluctuations.…”

Concepts towards Functional Eukaryotic Microbial Biogeography in the Ocean

“…Methodological developments, such as the use of mock communities in each sample [91], represent a practical solution to counter sampling, sequencing, and bioinformatics processing biases and to make samples more comparable, but post-processing and analytical methods that address the compositionality of the data are also important [92]. In a recent publication, Cohen et al [93] compiled a number of studies and their metatranscriptomic workflows, allowing others to easily find suitable workflows for themselves. Moreover, the data need to be understandable to everyone.…”

Gene expression dynamics of natural assemblages of heterotrophic flagellates during bacterivory

“…These studies can benefit substantially from the generation of reference genomes of dominant marine species, and new genomes from uncultured protists have been recently obtained by metagenomics [ 8 – 10 ] and single-cell genomics [ 11 – 14 ]. Recent studies using metatranscriptomics on protist communities have broadened our knowledge on different topics [ 15 ] such as trophic strategies [ 16 ], diel and seasonal cycles [ 17 , 18 ], nutrient responses [ 19 , 20 ], or functional biogeography [ 21 ]. The application of these tools to study bacterivory in the ocean is becoming very promising.…”