ITS2 metabarcoding of fungi from roots of trembling aspen
DNA was isolated from soil and trembling aspen root samples with the MoBio PowerSoil DNA Isolation Kit. The V3-V4 region of the bacterial 16S rRNA gene was amplified using the primer set 341F (5'-CCTACGGGNGGCWGCAG-3') / 805R (5'-GACTACHVGGGTATCTAATCC-3') 86. Primers contained the required Illumina adaptors at the 5' end of the primer sequences (5'-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3' for the forward primer and 5'-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3' for the reverse primer). Unique codes were added to each sample using the Nextera XT DNA Library Prep Kit. Indexed amplicons were purified using Agencourt AMPure XP according to the protocol in Illumina's "16S Metagenomic Sequencing Library Preparation" guide (Part # 15044223 Rev. B) and combined in an equimolar ratio. Paired-end sequencing (2 x 150 bp) of the 16S pools was carried out on an Illumina MiSeq sequencer at the National Research Council in Montreal.
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