ITS2 rDNA amplicon sequencing of the bark-inhabiting microbial community, twig of Olea europaea, sample P29, replicate 1
ITS2 rDNA amplicon was performed by PCR with the barcoded primers ITS3 (5'-GCA TCG ATG AAG AAC GCA GC-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3'). The PCRs were performed using the Q5 High-Fidelity DNA polymerase (BioLabs Inc.), they were run in 35 cycles and the conditions were: initial denaturation at 98 °C for 30 s, 98 °C denaturation for 10 s, 52° C amplification for 45 s and 72 °C elongation for 1 min, with a final 72 °C extension for 2 min. The PCR product was purified with SPRI AMPure XP paramagnetic beads (Beckman Coulter), pooled equimolarly and sent for library preparation and sequencing to Fasteris (Plan-les-Ouates, Switzerland). Sequencing was performed on the Illumina MiSeq platform with paired end mode (2 × 300 bp). Quality control of Illumina MiSeq paired-end reads was carried out using FastQC v. 0.11.8. Raw read pairs were filtered for average read quality threshold 26 using the script Reads_Quality_Length_distribution.pl (Báilint et al., 2014) and reads shorter than 150 bp were discarded. Paired-end assembly was performed using PANDAseq, followed by removing primer artifacts using the script python remove_multiprimer.py (Báilint et al., 2014). Paired-end reads were then reoriented in 5'-3' direction using fqgrep. Demultiplexing was carried out using the demultiplex.sh script (Báilint et al., 2014) modified to output fastq files. Sequencing adapters were removed from both ends of the sequences using the Fastx Toolkit.