title: 16S amplicon: apple twigs
design: The V4 region of the 16S SSU rRNA gene was amplified from undiluted samples in 35 cycles using primers 515F (5-GTGCCAGCMGCCGCGGTAA-3) and 806R (5-GGACTACHVGGGTWTCTAAT-3) also including Illumina adapters. 5M of mPNAs and pPNAs (PNAbio, USA) each were added to PCR mix. The PNA clamps mPNA and pPNA, block co-amplification of respectively plant mitochondrial and plastid targets in the samples and depending on amount of bacterial target in the sample, will subsequently reduce presence of plant reads in sequencing data. The amplification protocol for 16S consisted of an initial denaturation at 98C for 3 min, followed by amplification for 35 cycles at 95C for 30s , 75C for 10s for PNA annealing, 50C for 30s for primer annealing and extension at 72C for 30s, and a final extension of 1 min at 72 C.