title: 16S rRNA amplicon sequencing of Dactylorhiza traunsteineri rhizosphere microbiome - Soil - Replicate 1
design: Isolation of the metagenomic DNA was performed from the rhizosphere soil and the plantlet root wash lyophilized samples in a sterile environment. V3-V4 fragment of the 16S rDNA gene was amplified from the genomic DNA extracted from the soil and root samples (341F + adapter: ACACTCTTTCCCTACACGACGCTCTTCCGATCTNNNNCCTACGGGNGGCWGCAG; 805R + adapter: AGACGTGTGCTCTTCCGATCTGACTACHVGGGTATCTAATCC), in duplicates, using Q5 high-fidelity DNA polymerase and the following cycling conditions: 98C for 1 minute, followed by 20 cycles of 98C for 10 seconds, 62C for 30 seconds, 72C for 30 seconds, and a final extension at 72C for 2 minutes. The 16S rRNA gene copy concentrations in DNA extracts were determined through quantitative PCR and normalized to equal 16S rRNA fragment copy number to enhance comparability and reduce PCR bias, and purified using the Agencourt AMPure XP purification system. Sequencing indexes were introduced in a second amplification reaction with sample-specific barcodes (FWD: AATGATACGGCGACCACCGAGATCTACAC-[index]-ACACTCTTTCCCTACACGACG; REV: CAAGCAGAAGACGGCATACGAGAT-[index]-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT) using Q5 highfidelity DNA polymerase and the following cycling conditions: 98C for 1 minute, followed by 15 cycles of 98C for 10 seconds, 66C for 30 seconds, 72C for 30 seconds, and a final extension at 72C for 2 minutes. Amplicon libraries were purified using the Agencourt AMPure XP purification system and quantified using PicoGreen kit [ThermoFisher; Cat: P7589]. The libraries were pooled and diluted to 4 nM before being sequenced for 300 cycles pair-end on the Illumina MiSeq system using the MiSeq Reagent Kit v2.