Dataset: Diatom reference sequences
Deployment: LMG1401

Nine species of diatoms were isolated from the Western Antarctic Peninsula along the PalmerLTER sampling grid in 2013 and 2014. Isolations were performed using an Olympus CKX41 inverted microscope by single cell isolation with a micropipette (Anderson 2005). Diatom species were identified by morphological characterization and 18S rRNA gene (rDNA) sequencing. DNA was extracted with the DNeasy Plant Mini Kit according to the manufacturer’s protocols (Qiagen). Amplification of the nuclear 18S rDNA region was achieved with standard PCR protocols using eukaryotic-specific, universal 18S forward and reverse primers. Primer sequences were obtained from Medlin et al. (1982). The length of the region amplified is approximately 1800 base pairs (bp). Pseudo-nitzschia species are often difficult to identify by their 18S rDNA sequence, therefore, additional support of the taxonomic identification of P. subcurvata was provided through sequencing of the 18S-ITS1-5.8S regions. Amplification of this region was performed with the 18SF-euk and 5.8SR_euk primers of Hubbard et al. (2008). PCR products were purified using either QIAquick PCR Purification Kit (Qiagen) or ExoSAP-IT (Affymetrix) and sequenced by Sanger DNA sequencing (Genewiz). Sequences were edited using Geneious Pro software (http://www.geneious.com, Kearse et al., 2012) and BLASTn sequence homology searches were performed against the NCBI nucleotide non-redundant (nr) database to determine species with a cutoff identity of 98%.

BUSCO (Benchmarking Universal Single-Copy Orthologs) was used to assess the completeness of genomes and transcriptomes based on sets of single copy orthologous groups derived from OrthoDB that are highly conserved within multiple lineages (Felipe et al. 2015). Completed, duplicated and fragmented orthologs were determined by meeting an ‘expected score’ and having aligned sequences within two standard deviations of the BUSCO gene’s length. A second metric of completeness was performed by evaluating conserved pathways, such as the ribosome and spliceosome, using the single-directional best-hit method in the KEGG Automatic Annotation Server (KAAS) (Moriya et al. 2007). Finally contiguity, was calculated at the 0.75 level as according to Martin and Wang (2011) with custom scripts.

For each transcriptome, unassembled sequence reads were aligned to the final Trinity assembly using Bowtie 2 (Langmead 2012). Mapped reads were normalized by the Reads per Kilobase per Million reads method (RPKM) (Mortazavi et al. 2008).

Gene biogeographical distributions - 20 genes of interest were selected in the study to investigate the molecular basis of iron and light limitation in polar diatoms. Reference sequences for each of these genes were obtained from the F. cylindrus and P. tricornutum JGI genome portals and T. pseudonana and T. oceanica NCBI and GenBank repositories. Reference sequences were identified in the transcriptomes by translated nucleotide homology searches (tBLASTn) with an e-value cutoff of <10-5. A reciprocal tBLASTn homology search was performed for each transcriptome against the KEGG GENES database, using the single-directional best-hit method in the KAAS online tool to ensure consistent gene annotations (Moriya et al. 2007).

Subsequently, reference sequences were identified in the MMETSP protein database by BLASTp (e-value <10-5) homology searches among the diatom transcriptomes. The transcriptomes and their associated latitude and longitude were obtained from iMicrobe Data Commons (Project Code CAM_P_0001000) and the National Center for Marine Algae and Microbiota (NCMA). Custom Matlab scripts allowed global biogeographical distribution of key genes of interest to be mapped.