DNA Extraction and Amplicon Sequencing Protocol

DNA was extracted using a modified protocol of the Qiagen DNeasy plant kit (80 (link)), quantified using the QuBit double-stranded DNA (dsDNA) high-sensitivity assay (Life Technologies, Grand Island, NY). For amplicon sequencing, DNA was diluted with Tris-EDTA (TE; pH 8) to 1 ng · μL⁻¹, with V1-V2 16S rRNA amplicon sequencing and PCRs performed as described previously (34 (link)). Each reaction mixture included 5 ng template, 5 μL 10× buffer, 1 U Hi-Fi Taq, 1.6 μL 50 mM MgSO₄ (Life Technologies), and 200 nM (each) forward primer 27F_ill 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGagrgttygatymtggctcag‐3′ and reverse primer 338RPL_ill 5′‐GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGgcwgccwcccgtaggwgt‐3′; capital letters represent Illumina linker sequences on the 27F/338R primer pair (81 (link)). Reactions were cycled for 2 min at 94°C, 15 sec at 94°C (30 times), 30 sec at 55°C, 1 min at 68°C, and 7 min 68°C (for elongation). Purification was done using the MinElute kit (Qiagen, Valencia, CA), and product presence and removal of primer-dimers were verified by electrophoresis. V9 18S rRNA amplicons were generated as described above using the 1389F/1510R primer pair (24 (link)). Samples were sequenced using Illumina MiSeq v3 chemistry.