Picogreen assay

Polymerase chain reaction (PCR) was used to amplify the V5-8 hypervariable region of 16S rRNA genes for multiplex-pyrosequencing. A set of 10-nt barcodes designed by Roche was incorporated to the 926 Forward (5′-AAACTYAAAKGAATTGACGG) and 1392 Reverse (5′-ACGGGCGGTGTGTRC) primers for multiplexing (Supplementary Table S4) (DeAngelis et al., 2012 (link)). PCR reactions were performed using Taq DNA polymerase in 2X PCR Master Mix (Thermo Scientific, MA, USA) with the following conditions: (i) initial denaturation at 95°C for 3 min and (ii) 35 cycles of 95°C for 30 s, annealing at 55°C for 45 s, and elongation at 72°C for 90 s, followed by (iii) a final extension at 72°C for 10 min. Amplicons were purified using the GeneJET PCR Purification Kit (Thermo Scientific, MA, USA) and quantified using agarose electrophoresis and a Nanodrop 2000 spectrophotometer (Supplementary Table S4), before being sent to the Génome Québec Innovation Centre for precise quantification using PicoGreen assay (Thermo Scientific, MA, USA) and sequencing using a 454 GS FLX platform (454 Life Sciences – a Roche Company, Branford, CT, USA).

The V4 hypervariable region of the 16S rRNA genes was amplified using primers 515F and 806R (Apprill et al., 2015 (link); Parada et al., 2016 (link)). PCR was performed using high fidelity AccuPrime^TMTaq DNA Polymerase (Invitrogen, Cat. No. 12346086), including 35 cycles with an annealing temperature of 50°C. PCR clean-up and removal of small fragments was done with the Nucleo Spin Gel and PCR Clean-up Kit (Macherey-Nagel, Cat. No. 740609.250). Quantification of extracted PCR products was performed using PicoGreen assay (QuantIT, Thermo Fisher Scientific, Cat. No. P11496). Thereafter, pooling and sequencing of sample-specific libraries were performed by following the Illumina MiSeq protocol. Data processing was done using the IMNGS platform (Lagkouvardos et al., 2016 (link)), applying the UPARSE analysis pipeline (Edgar, 2013 (link)) with the following settings: Number of allowed mismatches in the barcode: 1, Min fastq quality score for trimming of unpaired reads: 20, Max rate of expected errors in paired sequences: 2, Minimum relative abundance of Operational Taxonomic Units (OTU) cutoff (0-1): 0.25%. The taxonomy of OTUs clustered at 97% sequence identity was determined using SILVA¹ (Pruesse et al., 2012 (link)). The data were submitted to the Sequence Read Archive and are available under the accession number PRJNA514431.

Single cell libraries were prepared according to the Smart-Seq2-protocol (22 (link), 23 (link)) with the following modifications. Pre-amplification PCR cycles were increased to 23 to obtain sufficient amounts of cDNA for the sequencing analysis. Primer dimers were eliminated by two 0.8x Ampure-XP bead clean ups. 0.3–0.5 ng of pre-amplified cDNA were subjected to library preparation with the Nextera XT library preparation kit, Illumina, San Diego, CA, USA in 8 μl reaction volume. Barcoded libraries were pooled and sequenced the with a S1 flow cell and a 300-cycle kit on a NovaSeq Illumina platform to obtain 150-bp paired-end reads. Quality controls were performed using TapeStation with D5000 high sensitivity tapes (both from Agilent, Santa Clara, CA, USA). Quantification was performed using Qubit high sensitivity kit (Thermo Fisher Scientific) after PCR preamplification, and the PicoGreen assay (Thermo Fisher Scientific) after Nextera XT had been performed.

Soil samples were briefly thawed and homogenised by wet sieving through pre-sterilized 2-mm mesh screens. Soil processing was carried out in a cold room (4 °C). Sieved samples (0.25 g) were washed (3 times) using 1X sterile Phosphate Buffered solution in a ratio 1:4 w/v.
DNA was extracted using the PowerSoil DNA Isolation kit according to manufacturer’s instructions (MoBio Laboratories Inc., Carlsbad, CA). DNA integrity was checked on a 3% agarose gel, and its quantity and quality were assessed using Qubit 2.0 fluorometer (Thermo Fisher Scientific), Nanodrop 2000 (Thermo Scientific) and Picogreen assay (Thermo Fisher Scientific).
The V4 region of the 16S rRNA gene was amplified using the PCR protocol developed by the Earth Microbiome Project (http://press.igsb.anl.gov/earthmicrobiome/emp-standard-protocols/16s/)^{77 (link)}. This protocol was modified such that the 12 base barcode sequence was included in the forward primer. Amplicons were sequenced on an Illumina using the 500 cycle MiSeq Reagent Kit v2 (http://www.illumina.com/) according to manufacturer’s instructions. In the sequencing process, PhiX in a low concentration spike-in was used to monitor sequencing quality control. Controls without template, as well as positive ones, were included to ensure amplicon generation was not contaminated during the protocol.