Metatranscriptomic Sequencing of Winter and Spring Planktonic Communities from Lake Erie, a Laurentian Great Lake

Lake Erie’s winter phytoplankton blooms have been documented for decades (1), and recent studies have revealed that these communities are dominated by centric, colonial diatoms, including Aulacoseira islandica and Stephanodiscus binderanus (2–4). However, the ecophysiology of these blooms and comprehensive analyses of the winter community have received limited attention (5). Here, we report 28 winter and 51 spring Lake Erie metatranscriptomes collected across spatial, temporal, and climatic gradients in an effort to address these knowledge gaps.

Opportunistic samples were collected by U.S. Coast Guard Cutter Neah Bay between February and March in 2019 and 2020 (6). Additional spring samples were collected in May and June of these same years by Canadian Coast Guard Ship (CCGS) Limnos and M/V Orange Apex, respectively. Sampling occurred in both the western and central basins of Lake Erie. Water column parameters were recorded prior to each sampling, along with meteorological conditions and ice cover. The samples were additionally analyzed for nutrient concentrations and phytoplankton taxonomy (7). Water column samples and plankton net concentrated samples were immediately processed onboard ship. Briefly, the samples were filtered through 0.22-μm nominal pore-size filters, flash-frozen, and stored at −80°C until extraction. RNA was extracted using standard phenol-chloroform methods with ethanol precipitation (8). Remaining DNA in samples was digested via a modified version of the Turbo DNase protocol using the Turbo DNA-free kit (Ambion). The samples were determined to be DNA-free via the absence of a band in the agarose gel after PCR amplification (initial denaturation at 95°C for 5 min, denaturation at 95°C for 45 s, annealing at 50°C for 45 s, and elongation at 72°C for 30 s; then repeat steps 2 through 4 for 30 cycles; and final elongation at 72°C for 10 min) using ⁵¹⁹F/⁷⁸⁵R 16S rRNA primers (⁵¹⁹F-CAG-CMG-CCG-CGG-TAA and ⁷⁸⁵R-TAC-NVG-GGT-ATC-TAA-TCC) with Escherichia coli K-12 MG1655 DNA as the positive control. The samples were quantified using the Qubit RNA HS assay kit (Invitrogen) and sent to the Department of Energy Joint Genome Institute for rRNA reduction and sequencing using an Illumina NovaSeq S4 2 × 151-nucleotide indexed run protocol (~15 million, 150-bp paired-end reads/sample).

The reads were filtered using BBDuk (v38.92) (9) to remove (i) contaminants, (ii) adapters, (iii) homopolymers of Gs of size 5 or more, (iv) right read segments where quality was 0, (v) reads with N bases, and (vi) reads with an average quality score less than 10, or minimum length of ≤51 bp or 33% of the full length. BBMap (v38.86) (9) removed contaminants (93% identity) and rRNA. Filtered reads were assembled using MEGAHIT (version 1.2.9) (–k-list 23, 43, 63, 83, 103, and 123) (10). The reads were then mapped to assembled contigs using BBMap (ambiguous = random). The assemblies were annotated using the IMG Annotation Pipeline (version 5.0.25) (11).

Taxonomic annotation of protein coding sequences (CDS) by IMG confirmed diatoms were a transcriptionally active component of the winter microbiome in this Laurentian Great Lake. CDS genes annotated as classes Coscinodiscophyceae (centric) and Bacillariophyceae (raphid, pennate) were highly represented relative to other photosynthetic eukaryotes (Table 1). Considering that winters of 2019 (near-maximum ice) and 2020 (negligible ice) spanned extremes in ice cover for Lake Erie, these metatranscriptomes offer a unique opportunity to investigate the influence of climate change on freshwater winter communities.

This work was supported by National Institutes of Health, NIEHS grant 1P01ES02328939-01, National Science Foundation grant OCE-1840715 (G.S.B., R.M.L.M., J.D.C., and S.W.W.), NSERC grant RGPIN-2019-03943 (R.M.L.M.), and JGI project 503851 (S.W.W. and R.M.L.M.). The work conducted by the U.S. Department of Energy Joint Genome Institute, a Department of Energy Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under contract DE-AC02-05CH11231.

We declare no conflict of interest.