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.