16s amplicon sequencing of Cetradonia linearis: whole thallus
The V4 variable region of the 16S rRNA gene was amplified using the 515F/806R primer pair, using HotStarTaq Plus Master Mix Kit (Qiagen, USA). The thermal profile included an initial denaturation at 94C for 3 minutes, followed by 28 cycles of denaturation at 94C for 30 seconds, annealing at 53C for 40 seconds, and extension at 72C for 1 minute, with a final extension at 72C for 5 minutes. Amplicons were pooled at equimolar concentration and purified using Ampure XP Beads. The purified PCR products were subsequently prepared for sequencing following the Illumina TruSeq DNA library preparation protocol. Sequencing was performed on the Illumina MiSeq platform at Molecular Research LP (Shallowater, TX).