IMIS - Marine Research Groups

Two liters of surface (5 m depth) seawater was initially filtered through 100 μm mesh to remove particulate matter, after which microbial biomass was collected by filtration through 0.22 μm Polyethersulfone (PES) membrane (Pall Corporation). The filters were immersed in RNALater (Qiagen) and stored at −20°C.
Seawater samples were collected at a near‐shore site on the northeast coast of PEI at 46°36.415′S; 37°58.553′E during the austral autumn (April) and winter (July) seasons in 2012 as well as the austral autumn (May) for years 2013, 2014, and 2015.
Method step description:

1. Genomic DNA (gDNA) and RNA were extracted from PES filters using the AllPrep DNA/RNA Mini Kit (Qiagen) according to the manufacturer's instructions. For the rRNA sequencing, isolated RNA was converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer's protocol. For the analysis of bacterial community composition, the V4‐V5 variable regions of the bacterial 16S rRNA gene were amplified by PCR using the E517F (5′‐CAG CAG CCG CGG TAA‐3′) and E969‐984R (5′‐GTA AGG TTC YTC GCG T‐3′) primers) with suitable multiplex identifier tags and sequencing primer binding sites attache. For the analysis of eukaryotic phytoplankton community diversity, PCR amplification of 18S rRNA gene sequences was carried out using primers 1391F: 5′‐GTA CAC ACC GCC CGT C‐3′ (Saccharomyces cerevisiae position 1629–1644) and EukB: 5′‐TGA TCC TTC TGC AGG TTC ACC TAC‐3′ (S. cerevisiae position 1774–1797) targeting the V9 regions of the eukaryotic SSU rRNA. Archaeal 16S rRNA gene sequences (V4‐V5 variable regions) were amplified using pr514–528: 5′‐GGT GYC AGC CGC CGC‐3′ and A906R: 5’‐CCC GCC AAT TCC TTT AAG TTTC‐3, respectively.
2. PCR amplification of the bacteria, phytoplankton and archaeal gene fragments was carried out in a 25 μL reaction volume comprising 10 ng of the extracted DNA and using KAPAHiFi Hotstart DNA Polymerase (KAPA Biosystems) according to the manufacturer's instructions. For bacterial 16S rRNA gene amplification, the reaction mixtures were subjected to the reaction conditions described in Matcher et al. (2011). For 18S rRNA amplification, the PCR cycling parameters were as follows: 98°C (45 s), 57°C (30 s), 72°C (45 s) for five cycles, 98°C (45 s), 65°C (30 s), and 72°C (45 s) for 15 cycles and a final extension at 72°C for 5 min. For archaeal 16S rRNA gene amplification, cycling conditions were used as for the 18S rRNA amplification with amendments of the annealing temperatures to 56°C (30 s) for the first five cycles and 59°C (30 s) for next 15 cycles.
3. The PCR amplification products were gel‐purified using the ISOLATE II PCR and Gel Kit (Bioline), subjected to emulsion PCR, and then sequenced using the GS Junior Titanium Sequencer (454 Life Sciences, Roche).