Metabarcoding of tree caulosphere
The polymerase chain reaction (PCR) amplification followed a two-step phased amplicon sequencing approach. In the initial step, two subsamples from each extraction were amplified using the ITS1F and ITS2 primer combination to target the ITS1 region (Gardes & Bruns, 1993). Primers included the published sequences along with one, two, three, four, or five random nucleotides as spacers, and complementary stubs for Illumina adapter pairing. This phased strategy effectively addresses the challenge of low diversity in the early cycles of Illumina sequencing by introducing a sequencing frame shift due to variable primer lengths, resulting in increased base diversity. This method enhances base read quality, boosts raw sequence output by approximately 15% (eliminating the need to add PhiX to the sequencing run due to phasing), and reduces sequencing errors (Wu et al., 2015). The PCR cycling conditions consisted of an initial 2-minute hot-start at 95C, followed by 35 cycles of 30 seconds at 95C for denaturation, 30 seconds at 50C (for ITS-1) or 55C (for ITS-2) for primer annealing, and 1 minute at 72C for primer extension, concluding with a final extension of 5 minutes at 72C. The PCR products were validated using gel electrophoresis on 1% agarose gels and visualized with UV light. In the subsequent step, Illumina adapters and barcodes were attached to the PCR products from the first phase. The Vincent J. Coates Genomics Sequencing Laboratory at the California Institute for Quantitative Biosciences (QB3), University of California, Berkeley, supplied dual-indexed adapters from their collection of 960 compatible dual-index pairs. The PCR protocol for this step began with a 2-minute hot-start at 95C, followed by 15 cycles of 30 seconds at 95C for denaturation, 30 seconds at 58C for primer annealing, and 50 seconds at 72C for primer extension, concluding with a final extension of 10 minutes at 72C. Clean-up after each PCR amplification was performed using AMPure XP beads in a 1.8 ratio. Quantification of PCR products was carried out using a NanoDrop 2000 spectrophotometer, and samples were normalized through dilution in PCR water. Fragment analysis of normalized pools was conducted using an Agilent Bioanalyzer
SUB15047970
MMGJ01-45