Dataset: Fe_Vit12_MetaT_Co-Limitation_RossSea_2014
Deployment: helicopter_Allen

Near-surface water samples were collected by helicopter in the Ross Sea near McMurdo Station, Antarctica.

Methodology: From Bertrand, E.M., McCrow, J.P., Moustafa, A., Zheng, H., McQuaid, J., Delmont, T., Post, A.F., Sipler, R., Spackeen, J.L., Xu, K., Bronk, D.A., Hutchins, D.A., Allen, A.E. (2015) Phytoplanktont-bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge. Proceedings of the National Academy of Sciences. 10.1073/pnas.1501615112

On January 16, 2013, seawater was collected from3mdepth at the sea ice edge in McMurdo Sound of the Ross Sea (77° 36.999' S 165° 28.464' E), using trace metal clean technique. Triplicate bottles (2.7 L) of each treatment (unamended control, + 1 nM added FeCl3, + 200 pM added cyanocobalamin, and + 200 pM cyanocobalamin and 1 nM Fe) were placed in an indoor incubator at 0 °C, ~45 µmol photonsm-2·s-1 of constant light. After 24 h, RNA sampleswere collected (450 mL) and nitrate uptake and primary productivity rates were measured. Samples were taken for Chl a at 0, 24, and 96 h. RNA was extracted using the TRIzol reagent (Life Technologies). Ribosomal RNA was removed with Ribo-Zero Magnetic kits, and the resulting mRNA enrichment was purified and subjected to amplification and cDNA synthesis, using the Ovation RNA-Seq System V2 (NuGEN). One microgram of the resulting high-quality cDNA pool was fragmented to a mean length of 200 bp, and Truseq (Illumina) libraries were prepared and subjected to paired-end sequencing via Illumina HiSeq. Reads were trimmed and filtered, contigs were assembled in CLC Assembly Cell (CLCbio), and ORFs were predicted (41). ORFs were annotated de novo for function via KEGG, KO, KOG, Pfam, and TigrFam assignments. Taxonomic classification was assigned to each ORF using a reference dataset, as described in the SI Appendix (Bertrand, 2015), and the Lineage Probability Index (LPI, as calculated in ref. 18). edgeR was used to assign normalized fold change and determine which ORFs were significantly differentially expressed in pairwise comparisons between treatments, considering triplicates, within a given phylogenetic grouping (42). For Fig. 2, diatom ORFs [identified as diatom via LPI analyses; LPI > 0.8 (18)] were clustered using MCL (Markov cluster algorithm) (21), and these clusters were used to produce MANTA plots (20). For Oceanospirillaceae ASP10-02a genome assembly, water was collected from 10 m in the Amundsen Sea on December 19, 2010. Metagenomic libraries were created with the OVATION ultralow kit (NuGen). Overlapping and gapped metagenomic DNA libraries were prepared for sequencing on a HiSeq platform (Illumina). CLC was used to assemble scaffolds, tetranucleotide frequencies of scaffolds were analyzed (43), and draft genomes were generated via binning scaffolds clustered (hierarchical) together in well-supported clades, refined using GC content and taxonomical affiliation (44). ORFs identified in metatranscriptomic analyses were mapped to the Oceanospirillaceae ASP10-02a genome bin from the best-scoring nucleotide alignment, using BWA-MEM with default parameters (45). ORFs with >99%similarity to the genome scaffold sequences were used for subsequent analyses of gene expression patterns within this population. Complete materials and methods are given in the SI Appendix (Bertrand, 2015).