Up to ten haphazardly selected trees per site were selected for micro-core sampling, for a total of 115 samples across 48 sites. Core samples consisted of three combined micro-cores (using the Trephor tool; (Rossi et al., 2006), taken from opposing sides of the trunk from asymptomatic tissue. Where symptomatic bleeds on the trunk were present, a second micro-core sample was taken directly from the wounds (three cores combined). These samples were chosen to represent asymptomatic trees (˜1/3), asymptomatic samples from symptomatic trees (˜1/3), and symptomatic samples from symptomatic trees (˜1/3) and to represent a broad geographical spread.   Initial quality control for DNA concentration was run using the Qubit High Sensitivity assay, after which libraries were prepared for MiSeq analysis. For amplification of the V4 region of the 16S rDNA gene, the universal primer set AATGATACGGCGACCACCGAGATCTACAC TATGGTAATT GT GTGYCAGCMGCCGCGGTAA (5' --- 3') and CAAGCAGAAGACGGCATACGAGAT NNNNNNNNNNNN AGTCAGTCAG CC GGACTACNVGGGTWTCTAAT (3'--5') was used. These primers included adaptor sequences used in MiSeq (the reverse primer contains a 12 base sequence (indicated by N’s) which allow for multiplexing of the samples). PCR mixtures contained 1µl of each forward and reverse primer (at 10µM concentrations), 2µl of template DNA, 0.25µl of Hot start Taq Polymerase (Qiagen), 1µl of dNTPs, 5µl of Q solution (part of the HotStart kit from Qiagen kit No: 203203), and 2.5µl of 10x PCR buffer in a final volume of 25µl. Thermal cycling consisted of an initial denaturation step at 95 °C for 15min, followed by 35 cycles of denaturation at 94°C for 45s, annealing at 50°C for 60s and extension at 72°C for 90s, with a final extension step at 72 °C for 10min. PCR amplifications were carried out in PTC-225Thermal Cycler (MJ Research). PCR products were run on a 2% agarose E-gel to check for bands at the expected range of between 300 and 350 bp and samples were cleaned using the Zymo DNA 96 Clean and Concentrator kit (Zymo Research). Quality control was finalized by running samples on the Shimadzu MultiNA to check the size and distribution of the libraries.
16S_Illumina_MiSeq_V86
SUB2124964
SOL5200_S17_L001_R1_001.fastq.SOL5200