Library preparation and sequencing
Approximately 250 to 500 mg of freeze-dried soil was used for total DNA
extraction using a NucleoSpin®Soil kit (Macherey-Nagel, Düren, Germany).
DNA concentration and purity of extracts were measured using a NanoDrop
2000 (Thermo Fisher Science, Wilmington, USA), and concentrations ranged
from 90 to 320 ng/µl. The entire ITS and partial LSU regions of the rDNA
operon were amplified using a modified ITS1 (5´–TCCGTAGGTGAACCTGC–3´)
(White et al., 1990), in which the two GG nucleotides from the 3´end
were removed compared to the original ITS1 primer, and LR5
(5’–TCCTGAGGGAAACTTCG–3’) (Vilgalys & Hester, 1990) were used as
forward and reverse primers, respectively. These primers were selected
because they amplify most known fungi (Tedersoo et al., 2015) and had no
known mismatches to most available sequences in Glomeromycota (Krüger et
al., 2012). In addition, the primers capture a wide range of non-fungal
microeukaryotes. Barcodes added to forward and reverse primers were
combined in sample-specific barcode pairs for multiplexed sequencing
(Table S3). Each 40 µl PCR reaction contained 20.4 µl nuclease free
water, 0.4 µl Phusion High-Fidelity DNA Polymerase (Thermo Fisher
Scientific, Hudson, NH, US), 8 µl 5× buffer, 500 µM of each primer, 200
µM dNTP mix, 2 µl DMSO, and 4 µl DNA template. The thermal cycle
protocol used was a 10 min initial denaturation at 95°C, 25 cycles of
denaturation (45 sec, 95°C), annealing (45 sec, 59°C) and elongation (90
sec, 72°C), and a final elongation (72°C, 10 min). PCR products were
visualized by gel electrophoresis. The resulting amplicons
(approximately 1,500 bp long) were purified with the ZR-96 DNA clean up
kit (Zymo Research, Irvin, California). The amount of PCR products used
for the pooled library was estimated to approximate equimolar amounts
based on observed electrophoresis band intensity. The pooled library was
sequenced together with root samples from the site at SciLifeLab/NGI
(Uppsala, Sweden) with six SMRT cells on the PacBio RSII sequencing
platform. Raw demultiplexed reads for the current study are available in
ENA (accession number: PRJEB47280).