Molecular analyses
In a dedicated room, we mixed the 15 g of soil with 20 ml of phosphate
buffer for 15 min as described in Taberlet, Coissac, Pompanon,
Brochmann, & Willerslev (2012); then we extracted eDNA using the
NucleoSpin® Soil Mini Kit (Macherey-Nagel, Germany) with a final elution
in 150 μl for both soil samples and with one negative extraction control
every 23 samples (total: 12).
We amplified eDNA of bacteria, eukaryotes, fungi, springtails, insects
and earthworms using primers designed for markers Bact02 (Bacteria and
Archaea: Taberlet et al., 2018), Euka02 (Eukaryota: Guardiola et al.,
2015), Fung02 (Mycota: Epp et al., 2012; Taberlet et al., 2018), Coll01
(Collembola, i.e. springtails: Janssen et al., 2018), Inse01 (Insecta:
Taberlet et al., 2018), and Olig01 (Oligochaeta, i.e. earthworms:
Bienert et al., 2012; Taberlet et al., 2018). We selected this set of
markers to cover a wide range of organisms at different taxonomic
resolution as we included three generalist markers (targeting entire
superkingdoms or kingdoms: Bact02, Euka02 and Fung02) and three more
specific markers (targeting from classes to subclasses: Coll01, Inse01,
Olig01). All these markers are well suited for metabarcoding analyses
thanks to the low number of mismatches in the priming regions across
target organisms, and they perform well with potentially degraded DNA
due to the relatively short length of amplified fragments (Taberlet et
al., 2018; Table S1). We used forward and reverse primers tagged on the
5’-end with eight-nucleotide long tags with at least five nucleotide
differences among them (Coissac, 2012) and combined them in a way that
all PCR replicates were represented by a unique combination of forward
and reverse tags. This allowed us to uniquely identify each PCR
replicate after sequencing. We randomized all samples on 96-well plates
and included 24 bioinformatic blanks, 12 PCR negative controls and one
PCR positive control. The positive control was constituted of genomic
DNA of eight bacterial and two fungal strains (i.e., Pseudomonas
aeruginosa, Escherichia coli, Salmonella enterica, Lactobacillus
fermentum, Enterococcus faecalis, Staphylococcus aureus, Listeria
monocytogenes, Bacillus subtilis, Saccharomyces cerevisiae, Cryptococcus
neoformans ) at known concentrations (ZymoBIOMICS™ Microbial Community
DNA Standard II, Zymo Research, USA; 1:10 diluted) and we used it to
check for potential cross-contaminations and to monitor amplification
and sequencing performances.
We determined the optimal number of amplification cycles and DNA
dilution by conducting a qPCR essay on 48 randomly selected samples,
using 1 μl of 1:1000 diluted SYBR® Green I nucleic acid gel stain
(Invitrogen™, USA), and both undiluted and 1:10 diluted DNA, with a
real-time PCR thermal cycler set to standard mode. This step is useful
to avoid over-amplifying eDNA and to limit chimera formation.
Based on qPCRs results, we finally performed 42 (Bact02), 45 (Euka02,
Fung02) or 55 (Coll01, Inse01, Olig01) amplification cycles on diluted
(Euka02, Coll01) or undiluted (Bact02, Fung02, Inse01, Olig01) DNA.
Amplification consisted of 20-μl reactions with 10 μl of AmpliTaq Gold
360 Master Mix 2X (Applied Biosystems™, Foster City, CA, USA), 2 μl of
forward and reverse primer mix (initial concentration of each primer: 5
μM), 0.16 μl of Bovine Serum Albumin (i.e. 3.2 μg; Roche Diagnostic,
Basel, Switzerland) and 2 μl of eDNA. We performed reactions in 384-well
plates, with four PCR replicates per sample (Gentile F. Ficetola et al.,
2015), setting the following PCR profiles: an initial step of 10 min at
95°C; several cycles of a 30 s denaturation at 95°C; a 30 s annealing at
53°C (Bact02), 45°C (Euka02), 56°C (Fung02), 51°C (Coll01), 55°C
(Olig01) or 52°C (Inse01); a 90 s elongation for Bact02 and Fung02, or a
60 s elongation for all the others markers at 72°C; a final elongation
at 72°C for 7 min. After amplification, we pooled together all amplicons
of the same marker and visualized a 5-μl aliquot by high-resolution
capillary electrophoresis (QIAxcel Advanced System, QIAGEN, GERMANY) in
order to check fragments length and monitor primer dimers. Finally, for
each marker, we purified six subsamples of the pooled amplicons
separately, using the MinElute PCR Purification Kit (QIAGEN, GERMANY) as
per the manufacturer’s instructions and combined them again. Libraries
were prepared following the MetaFast protocol (Taberlet et al., 2018)
and sequenced using the MiSeq (Bact02 and Fung02) or HiSeq 2500 (all
others) Illumina platforms (Illumina, San Diego, CA, USA) with a
paired-end approach (2 × 250 bp for Bact02 and Fung02, and 2 × 150 bp
for the others markers) at Fasteris (SA, Geneva, Switzerland).