IMIS

Surface water samples (5 m depth) were collected with 5 L Niskin bottles from 17 different locations. Each location was assigned to one of the following four categories: Collins Glacier (‘C’ stations), Nelson Glacier (‘N’ stations), Fildes Bay (‘F’ stations) and Inner Bay (‘IB’ stations). Seawater samples (5 L) were prefiltered on board through 100 μm pore mesh and stored in acid-washed carboys and kept on dark until subsampling at the laboratory.
Study Extent: Water samples were taken at Fildes Bay, King George Island, Antarctica, on 7 and 8 February 2012.
Method step description:

1. For photosynthetic eukaryote identification, plastidial 16S rRNA PCR products (in triplicates) were obtained using primer pair PLA491F–OXY1313, purified using Zymoclean kit (Zymo Research) and checked on an Agilent Bioanalzyer DNA1000 chip for the absence of primer dimers, and quantified using a PicoGreen dsDNA quantitation reagent (Invitrogen). Equal amount of purified PCR products were pooled for subsequent 454 pyrosequencing using a Roche GS-FLX Junior.
2. For bacterial identification, general 16S rRNA PCR products (in triplicates) were obtained using primers 515Fseq and 806rcbc (Caporaso et al.2011) following conditions from Earth Microbiome Project (EMP) (Gilbert, Jansson and Knight 2014). Illumina primer constructs were obtained from EMP also. Amplicons were quantified using KAPA Library Quantification Kit (KAPA Biosystem) and sequenced using Illumina Miseq following Caporaso et al. (2012) protocol. 12 pM of qPCR quantified amplicons pool were sequenced using a 300 cycles Illumina Miseq kit.