Ukiyo-e-Seq collects paired imaging and transcriptomic data from individual uni- and oligocellular marine organisms.

A. Overview of Ukiyo-e-Seq, starting from organismal enrichment with a plankton net (1, keep particulate) and later tea sieves and filter paper (2, keep middle fraction), followed by image-assisted picking (3) and single-well RNA-Seq (4). B. Images for 4 representative wells in brightfield (middle) and 1 additional representative well in brightfield and epifluorescence (bottom, emission wavelengths shown) for samples acquired near Coogee beach, NSW, Australia C. Cumulative distribution of sequencing coverage across 70 wells in two separate experiments. Four empty wells were used as negative controls. D. Schematic of the merge-split metatranscriptome assembly pipeline. E. Read coverage heatmap across organisms of the most abundant assembled contigs, showing both shared/public contigs (blocks) and private ones (vertical stripes).

Global and single-well taxonomic analysis of plankton transcriptomes.

A. Analytical workflow to compute taxonomic coverage maps for each well. B. Abundance treemap of taxonomic units. Taxa are numbered within each superkingdom by the number of reads mapping to that taxonomic unit (lower numbers indicate larger read counts). C, D: Paired images and phylogenetic trees on sequence contigs for two representative wells, containing a photosynthetic diatom (C) and a brown trout egg or larva (D). The size of each ball is proportional to the fraction of contigs assigned to that taxon by Kraken 2. Colours by superkingdom (green: eukaryotes, orange: bacteria, purple: viruses, grey: unassigned).

Functional and structural analysis of proteins and complexes from individual organisms.

A. Workflow of functional analysis of proteins found in individual wells. B. Presence of genes belonging to the KEGG photosynthesis pathway across four representative wells. C. Multiple sequence alignment of a newly identified D1-CG1 photosystem II reaction center protein D1 (PsbA) sequence identified in well K4 with three other PsbA sequences and corresponding pictures from [25]. D. Three-dimensional structural reconstruction of photosystem II from well K4. E. Distribution of expression for contigs assigned to Salmo trutta in well I5 (image in top left inset) and structural prediction of two predicted proteins from highly expressed contigs. Structural predictions via AlphaFold 3 [26].

Brightfield images (zoom in) of the plankton captured in each of the 66 wells during our two experimental batches.

Examples of wells with private contigs. No relationship between the presence of private contigs and sequencing depth was found.

Phylogenetic tree of all taxa identified by Kraken 2 across all contigs (all wells together). Branches were collapsed at the phylum level for simplicity and coloured by superkingdom.

Structural reconstruction of Photosystem I from well K4, the same as in Figure 3D.