title: ITS2 rDNA amplicon sequencing of the free-living rock-inhabiting green algal community - Balt49, replicate 2
design: ITS2 rDNA amplicon was performed by nested PCR with the primers 1378j02 (5-TTG CCT TGT CAG GTT GAT TCC-3) and ITS4 (5-TCC TCC GCT TAT TGA TAT GC-3) in the first step and barcoded 5.8F-Chlorophyta (5-GAA TTC CGT GAA CCA TCG AAT CTT T-3) and ITS4 primers in the second step. The PCRs were performed using the Q5 High-Fidelity DNA polymerase (BioLabs Inc.), they were run in 22 and 24 cycles in the first and second step, respectively 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 (Bilint 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 (Bilint et al., 2014). Paired-end reads were then reoriented in 5'-3' direction using fqgrep. Demultiplexing was carried out using the demultiplex.sh script (Bilint et al., 2014) modified to output fastq files. Sequencing adapters were removed from  both ends of the sequences using the Fastx Toolkit.