Molecular analysis
Sediment samples were centrifuged at 4000 rpm for 10 minutes, supernatant was removed and subsamples were mixed. Wet samples were weighted to 10 g (in the following referred to as metabarcoding treatment HTS 10) and 0.5 g (HTS 0.5), and DNA was extracted using DNeasy PowerMax Soil Kit and DNeasy PowerSoil Kit (Qiagen, Germany), respectively (three samples that were processed with PowerMax Kit had < 10 g input, see Table S1). Except for the amount of chemicals, these kits use identical chemistry and protocols. To enhance the cell lysis, we modified the initial step by adding Proteinase K (10 mg/ml) and 1M DTT (dithiothreitol) together with the C1 solution from the extraction kits. For the PowerMax Kit (10 g of sediments) 60 µl of Proteinase K and 100 µl of DTT, and for the PowerSoil Kit (0.5 g of sediments), 4 µl of Proteinase K and 25 µl of DTT was added, respectively; following overnight incubation at 56 ºC. For potentially higher DNA yield, the elution was performed twice by adding half of the recommended amount of the buffer onto a spin column membrane and incubated at room temperature for 3 minutes. The rest of the steps were performed following manufacturer’s instructions.
PCRs were performed using uniquely tagged primers rbcL-646F (5’-ATG CGT TGG AGA GAR CGT TTC-3’) and rbcL-998R (5’-GAT CAC CTT CTA ATT TAC CWA CAA CTG-3’), which amplify 331 base pairs (bp) of the large subunit of the ribulose-bisphosphate carboxylase/oxygenase (rbcL) gene (Kellyet al. 2018) (Table S2). We also tested the primers of Diat_rbcL_708F and R3 (Vasselon, Rimet, Tapolczai & Bouchez 2017), which amplify a shorter fragment (312 bp) of the same region. However, the PCR results were visually superior for rbcL-646F and rbcL-998R primer pair (data not shown), thus here, we decided to proceed only with the latter primers. The 25 µl PCR mix consisted of 5 µl of Hot Start FirePol Master Mix (Solis BioDyne, Estonia), 0.5 µl forward and reverse primer, and 1-3 µl of template DNA. The rest of the volume was filled with nuclease-free water. PCR conditions were as follows: 95 ºC for 15 minutes (hot start), 32-35 cycles of 95 ºC for 20 s, 55 ºC for 45 s, 72 ºC for 60 s, and final extension at 72 ºC for 5 minutes. Three replicate PCRs were performed per sample, following sample pooling and checking the yield of PCR products during gel electrophoresis by pipetting 5 µl PCR product on 1% agarose gel. Amplicons per sample were pooled as based on their relative quantity and purified using Favor-Prep™ Gel/PCR Purification Kit (Favorgen-Biotech Corp., Austria), following the manufacturer’s instructions. Steps of DNA extraction, PCR and sequencing included both negative and positive controls. Sample preparations, as well as DNA isolations, were conducted under laminar flow clean bench, using 30 min UV sterilization prior analyses. Sequencing was performed on the Illumina MiSeq (2x250) using MiSeq Reagent Kit v2.