Exploring hidden Phytophthora communities via environmental DNA massive metabarcoding
Amplicon libraries were generated using a nested PCR approach. In the first PCR round the Phytophthora-specific primers 18Ph2F and 5.8S-1R were used (Scibetta et al. 2012). For the second PCR round, fusion primers were designed following the GS Junior System Guidelines for Amplicon Experimental Design (Roche, 2010), selecting the unidirectional sequencing protocol for library construction (Lib-L chemistry for emPCR, ‘One-Way Reads’). Forward fusion barcoded primers were based on the universal ITS6 primer (Cooke et al., 2000) (5’-A-KEY-MID-ITS6-3’) and the reverse primer was the same reverse primer used in the first PCR round (5’-B-KEY-5.8S-1R-3’)