title: ITS amplicon sequencing soil
design: Amplicon sequencing targeting the 16S rRNA as well as the Internal transcribed spacer (ITS) region was used to determine the composition of the bacterial and fungal communities, while qPCR targeting the 16S rRNA gene was used to determine the quantity of bacteria in the soil. DNA was extracted from 0.22-0.29 g of soil from samples where enough soil was present (Supplementary Table 1) following the power soil pro kit protocol (Qiagen). Quantitative Polymerase Chain Reaction (qPCR) using an MX3005p qPCR instrument (Agilent, Santa Clara, CA, United States) was performed to quantify the amount of bacterial 16S rRNA gene copies. We targeted partial 16S rRNA gene sequence using universal primers Bac908F (5-AAC TCA AAK GAA TTG ACG GG-3) and Bac1075R (5-CAC GAG CTG ACG ACA RCC-3) (Ohkuma & Kudo, 1998). The qPCR mixture contained 2 l template DNA, 1 l of each primer (10 pmol l-1), 2 l BSA (10 ng l-1), 10 l LightCycler Mastermix (Roche) and 4 l dH2O to a final volume of 20 l. Samples were run in triplicate including triplicate negative qPCR controls without addition of template DNA. Samples were run on a Stratagene Mx3005P (Agilent technologies) with the following thermal conditions: 95 C for 5 min, 45 cycles with denaturation at 95 C for 30 s, annealing at 55 C for 30 s, elongation at 72 C for 15 s, followed by fluorescent acquisition 80 C at 5 s. A melting curve was produced using 1 cycle at 95 C at 30 s, 60 C at 1 min and 95 C at 30 s. The V3 and V4 of the 16S rRNA gene was amplified with primers Bac341F (5-CCT ACG GGN GGC WGC AG-3) and Bac805R (5-GAC TAC HVG GGT ATC TAA TCC-3). The 16S rRNA gene amplification was performed according to a modified Illumina protocol. The PCR mixture contained 2 template DNA, 2  KAPA HiFi HotStart polymerase (Kapa Biosystems, Inc., Wilmington, MA, United States), 0.2 M forward primer and 0.2 M reverse primer. The thermal cycling was run with an initial denaturation step at 95C for 3 min, 30 cycles with denaturation at 95C for 30 s, annealing at 55C for 30 s, elongation at 72C for 30 s and a final elongation at 72C for 5 min. For the ITS library preparation we used primers ITS3 (5- GATGAAGAACGYAGYRAA-3) and ITS4 (5-CTBTTVCCKCTTCACTCG-3) for amplification of the ITS2 region (Toju et al., 2012). The PCR mixture contained 10 l template DNA. Thermal conditions were set as follows: An initial denaturation step at 95 C for 3 min, followed by 22 cycles with denaturation at 95 C for 30 s, annealing at 56 C for 30 s, elongation at 72 C for 30 s, and a final elongation at 72 C for 5 min. The PCR products were cleaned using 30 l AMPure XP magnetic beads for both 16S and ITS amplicons. The second round of PCR was run with 2 l of the amplified and cleaned PCR product for 10 cycles to incorporate overhang adapters and was run with the same conditions as the previous PCR for 16S and ITS thermal conditions, respectively. Products were cleaned, and the Nextera XT Index primers were incorporated in a third PCR reaction which was run for 8 cycles following the previous condition and an annealing temperature of 55 C. The PCR products were quantified using a Quant-iT dsDNA BR assay kit on a FLUOstar Omega fluorometric microplate reader (BMG LABTECH, Ortenberg, Germany), diluted and pooled together in equimolar ratios. The pool was quantified using the Quant-iT dsDNA BR assay kit on a Qubit fluorometer (Thermo Fisher Scientific, Waltham MA) and then sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA) which produces two 300-bp long paired-end reads.