title: 16S rRNA gene  amplicon sequencing of wheat associated metagenome
design: High-throughput sequencing of the V3V4 region was performed by LGC, Biosearch Technologies on an Illumina MiSeq platform according to the standard protocols with PNA blocker.  Amplicons were generated using a two-step PCR protocol. Briefly, V3V4 region was amplified using 341F and 785R primers  that contained the adapter overhang nucleotide sequences for forward and reverse index primers. Amplicons were purified using AMPure XP beads (Beckman Coulter) and set up for the index PCR with Nextera XT index primers (Illumina). The indexed samples were purified using AMPure XP beads, quantified using the Fragment Analyzer (Advanced Analytical) and equal quantities from each sample were pooled.