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).