title: Fungal (ITS) microbiome of soil: rhizosphere soil
design: DNA was extracted from 25 mg fresh (frozen soil) with the DNeasy PowerSoil Pro Kit (Qiagen, Germany). Fungal community composition was determined by sequencing the ITS2 gene using primer pair ITS86F  ITS4 (Vancov and Keen, 2009). Sequencing library preparation involved two PCR steps. The first PCR reactions contained 2.5 L template DNA (5 ng L-1), 12.5 L KAPA HiFi HotStart ReadyMix (Roche, Ireland), and 0.5 L 1M forward and reverse adapted primers (Eurofins, UK).  PCR conditions for ITS were as follows: 3 minutes at 95C, then 25 cycles of: 30 seconds at 95C, 30 seconds at 54C, and 30 seconds at 72C, and finally five minutes at 72C. Because ITS DNA yields are lower, we included three PCR replicates for each experimental unit and pooled the products at this stage. . In a second PCR, Illumina sequencing adaptors were added on 16S rRNA and ITS amplicons using Nextera XT kit in a 50 L reaction: 5 L PCR 1 product, 5 L of each Nextera index primer, 25 L KAPA HiFi HotStart ReadyMix, and 10 L nucleic acid free water. The second PCR conditions were: 3 minutes at 95C, then 7 cycles of: 30 seconds at 95C, 30 seconds at 55C, 30 seconds at 72C, and finally 5 mins at 72C. Amplicons were again purified using Ampure XP beads (Beckmann Coulter, UK) according to manufacturers instructions, and eluted in 15 L TE buffer. Samples were sequenced on a Illumina MiSeq Standard Flow Cell, resulting in paired-end 300 base pair reads.