Table of Contents

• Coverage
• Dataset Description
  • Methods & Sampling
  • Data Processing Description
  • BCO-DMO Processing Description
• Related Publications
• Related Datasets
• Parameters
• Instruments
• Project Information
• Funding

This project required sequencing whole genomes and transcriptomes from single cells picked from two cultures. One was Schmidingerella arcuata (urn:lsid:marinespecies.org:taxname:732664), isolated from the East Coast of the United States in Long Island Sound; the other was Schmidingerella meunieri (urn:lsid:marinespecies.org:taxname:732864), isolated by S. Strom from Puget Sound on the West Coast of the United States. S. arcuata was collected from the surface waters of northeastern Long Island Sound, CT (41.31°N, 72.06°W), using a 20-micrometer (µm) mesh plankton net. Single cells were isolated with drawn capillaries and moved to six-well culture plates with 0.2-µm-filtered sample water. The goal was to quantify the degree of difference in genome architecture for these two close congeners.

The cultures were grown in filtered seawater at 18 degrees Celsius (°C) on a 12:12 light cycle and fed the dinoflagellate Heterocapsa triquetra and the prymnesiophyte Isochrysis galbana. Individual cells were picked with a drawn capillary pipette and processed for sequencing as detailed below (from Smith et al 2020).

The SMART-Seq2 v4 Ultra Low input RNA kit (Cat: 634889; Takara, Mountain View, CA) was used for whole transcriptome amplification (WTA) following the manufacturer's protocols, with the exception that we quartered the reaction volumes. For whole-genome amplification (WGA), the Repli-g single-cell kit (Cat: 150343; Qiagen, Hilden, Germany) was used following the manufacturer’s protocols. The products (cDNA for WTA, gDNA for WGA) were quantified with the dsDNA Qubit assay (Invitrogen, Waltham, MA) and polymerase chain reaction-checked with eukaryotic 18S rDNA and genus-specific ITS primers. Minimum bacterial contamination was confirmed by polymerase chain reaction with 16S rDNA primers Sequencing libraries were prepared with the Illumina Nextera XT kit (Cat: FC1311096; Illumina, San Diego, CA), then processed with Illumina HiSeq 2500 at Macrogen Sequencing (Geumcheon-gu, Seoul, South Korea).

The isolation/cultivation was done in 2014-2015. The sequencing work was all completed and analyzed by the suummer of 2020.

Raw reads from WTA and WGA sequencing were trimmed for quality and size (Q28 and minimum length of 200 and 1,500 bp, respectively) using BBMap (V38.39). After trimming, two single-cell WTAs were assembled together using rnaSPAdes (V3.13.1), and seven singe-cell WGAs were assembled together using both SPAdes (V3.13.1) and MEGAHIT (V1.2.9). The MEGAHIT genome assembly was used for the final analysis.

Assemblies were processed through custom python scripts (https://github.com/maurerax/KatzLab/tree/HTS-Processing-PhyloGenPipeline) for the removal of rDNA and prokaryotic transcripts, and for the identification of orthologous gene families using USEARCH (V9.2) with OrthoMCLdatabases (V2.0.9).

- Imported original file "NCBI_data_Schmidingerella.csv" into the BCO-DMO system.
- Removed "N" and "W" from the latitude and longitude columns.
- Made longitude negative to indicate the west direction.
- Renamed fields to comply with BCO-DMO naming conventions.
- Saved the final file as "924260_v1_ncbi_accessions.csv"

NSF Award Abstract:
Planktonic ciliates are key members of marine food webs where they serve diverse roles, including as food chain links between smaller microbes and larger plankton. Due to their small size and difficulties in identifying and cultivating them, we know less about ciliate diversity and distributions in the ocean than we do about larger organisms such as fish and invertebrates. Previous work from this team measured ciliate diversity in coastal waters and found that distinct genetic variants were separated in time and space in a way that could be related to factors such as ocean temperature, salinity, and depth gradients. Many questions remained unanswered, and it is important to understand the environmental factors that control the diversity and distribution of plankton such as ciliates to predict how these organisms may respond to a changing enviroment in the coming decades. This project focuses on: 1) how ciliate species are delineated using single-cell genomics and transcriptomics; 2) DNA-based studies of all ciliates and other planktonic members of the SAR clade (Stramenopila, Alveolata, Rhizaria), which will provide ecological context; 3) in situ gene expression by single-cell and meta- transcriptomics; and 4) laboratory studies of gene expression in cultivated ciliate species. This project involves training of postdoctoral scholars, graduate students, and undergraduates. The researchers are committed to creating diverse and inclusive research labs; recruitment of participants will be done through partnership with appropriate groups on our campuses. The project integrates with summer Research Experiences for Undergraduates (REU) activities at both Smith College and UCONN (including the UCONN/Mystic Aquarium joint REU), which are especially focused on underrepresented students. This project also enhances efforts to broaden understanding of biodiversity in partnership with the UCONN Noyce Scholars Program, which facilitates career-changing STEM professionals to become teachers in underserved secondary schools.

This project will assess distributions of reproductively-isolated species, determined using a new method to characterize regions of the ciliate germline genome. Furthermore, it will use phylogenomic methods to identify clade-specific transcripts (e.g. those of spirotrich ciliates) within metatranscriptomes from the shelf environment and to expand knowledge of ciliate function with single-cell transcriptomics of field-collected cells. These approaches will be a substantial improvement over the culture-based methods that are potentially biased towards "weedy" species in the ocean. The combination of definitive species identification with assessment of function via single-cell and meta- transcriptomics promises to provide significant advances in marine plankton ecology. The research focuses on two broad questions: 1) does the observed high diversity in phylogenetically-informative genes reflect reproductive isolation and functional differentiation in planktonic ciliates? and 2) do different co-occurring species of planktonic ciliates show substantial functional differences that correspond to different niches in the ocean? The project assesses species boundaries (i.e. reproductive isolation) through analyses of patterns in the germline micronuclei of planktonic ciliate morphospecies; characterizes transitions of closely-related ciliates across ecological gradients in the ocean; and examines functional differences within and between species, and in communities, through analyses of transcriptomics.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.