Intercomparison of metatranscriptomic methods for characterizing microbial eukaryote contributions to the biological carbon pump

Meta-eukomic: Intercomparison of metatranscriptomic methods for characterizing microbial eukaryote contributions to the biological carbon pump

Microeukaryotes are core components of the biological carbon pump through their roles in primary production, carbon export, and trophic transfer in the marine food web.

Metagenomics, metatranscriptomics, metabarcoding, and eDNA sampling are increasingly being performed as core measurements on individual cruises as well as being incorporated into large oceanographic surveys (e.g. Tara Oceans, Bio-GO-SHIP, BioGeoTraces). However, there is currently no single gold standard practice for the collection, preservation, or processing of these samples—nor is there a sense of how variable these measures might be among labs or research cruise endeavors. Intercomparison and intercalibration of ‘omic studies is a necessary next step for our field.

The goal of this activity is to determine the extent of variability in existing metatranscriptomic pipelines through a deliberate community-led intercomparison to build international confidence in methodological choices.

Fig. 2. Overview of the metatranscriptomic collection and analysis process. Our intercalibration exercise focuses on the steps highlighted in orange (extraction and bioinformatic analyses). Briefly, each participant was assigned at least two filter slices that will be used to test various comparisons. The intercomparison will begin at Step 2: RNA extraction, given that samples have already been collected using in-situ McLane pumps in Summer 2023 (see Fig. 1). We will also test the effects of poly-adenylated tail selection vs. rRNA depletion, with Andrew Allen (Scripps/JCVI) performing riboPOOL RNA depletions and the Columbia Genome Center performing the Ribozero protocol (Step 3: Sequence prep). Other participants will compare different sequencing facilities by sending one sample to the Columbia Genome Center (funded by H. Alexander), and the other to a lab-preferred sequencing center (e.g., University of Washington, Genewiz; self-funded) (Step 4: Sequencing). After sequences are obtained, all samples will be pooled and used with a consistent pipeline (Krinos et al. 2022) to determine the effects of Steps 1-4. In addition, users will be asked to process a designated sample using their own computational procedure to isolate sources of variation in the bioinformatic steps (Steps 5 & 6).

Figure caption: Overview of the metatranscriptomic collection and analysis process. This intercalibration exercise focuses on the steps highlighted in orange (extraction and bioinformatic analyses). Briefly, each participant was assigned at least two filter slices that will be used to test various comparisons. The intercomparison will begin at Step 2: RNA extraction, given that samples have already been collected using in-situ McLane pumps in Summer 2023. After sequences are obtained, all samples will be pooled and used with a consistent pipeline to determine the effects of Steps 1-4. In addition, users will be asked to process a designated sample using their own computational procedure to isolate sources of variation in the bioinformatic steps (Steps 5 & 6)

Planned Activities & Products:

  • Bimonthly Webinar: Our bimonthly webinar series is open to all - please give your input on themes, ideas and interest in speaking or recommend speakers (two per webinar) that span omic method topic areas.
  • Two in-person meetings to interpret and process generated metaT data, a Hackathon and a Synthesis Session (open application and invitations)
  • Bimonthly webinars with breakout sessions to engage with the broader microbial ‘omic scientific community (open to all)
  • A best practices “how-to” guide, to increase accessibility to non-experts, and to foster connections within the marine microbial ‘omic community, allowing for growth in our understanding of the strengths and limitations of this method.
  • Publication of intercomparison results

 

Capacity building

For in person meetings 10 early career researchers (late graduate students and postdoc scholars) will be selected from open applications – (criteria: graduate program of study and applicability to the intercomparison, interest level in bioinformatics, and career stage; emphasizing the inclusion of students with backgrounds traditionally underrepresented in STEM).

EC researchers will gain advanced computational biology training in the first meeting; EC ideas and intercomparison results will be included in the planned publication via their participation in the second meeting.

Read the full proposal

Apply to participate

Organizing Committee

Harriet Alexander (Woods Hole Oceanographic Institution)

Natalie Cohen (University of Georgia Skidaway Institute of Oceanography)

Sarah Hu (Texas A&M)

Adrian Marchetti (University of North Carolina at Chapel Hill)