2.3 PCR, library preparation and sequencing
The hypervariable region V3-V4 of the bacterial 16S rRNA gene was amplified with the PCR primer pairs 338F (5’-ACTCCTACGGGAGGCAGCAG-3’) and 806R (5’-GGACTACHVGGGTWTCTAAT-3’) (Mori et al., 2014). The fungal ITS1 region was amplified using PCR primers ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and ITS2R (5’-GCTGCGTTCTTCATCGATGC-3’) (Adams et al., 2013). PCR amplification was performed as follows: initial denaturation at 95 °C for 3 min, followed by 27 (338F_806R) / 35 (ITS1F_ITS2R) cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 72 °C for 45 s, single extension at 72 °C for 10 min, and termination at 4 °C. The PCR mixtures contained 5 × TransStart FastPfu buffer (4 μL), 2.5 mM dNTPs (2 μL), forward primer (5 μM; 0.8 μL), reverse primer (5 μM; 0.8 μL), TransStart FastPfu DNA Polymerase (0.4 μL), bovine serum albumin (BSA; 0.2 μL), template DNA (10 ng), and up to 20 μL ddH2O. PCR reactions were performed in triplicate. The PCR products were extracted from 2% agarose gel and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) according to manufacturer’s instructions and were quantified using Quantus™ Fluorometer (Promega, Madison, WI, USA).
Purified amplicons were pooled equimolar and were paired-end sequenced (2 ×300) on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database under Accession Number: PRJNA609016.
2.4 Sequence processing
Raw fastq files were quality-filtered by Trimmomatic (Bolger et al., 2014) and merged by FLASH (Magoc & Salzberg, 2011) applying the following criteria: (i) Reads were truncated at any site receiving an average quality score < 20 over a 50-bp sliding window. (ii) Sequences with overlap longer than 10 bp were merged according to their overlap with mismatch of no more than 2 bp. (iii) Sequences of each sample were separated according to barcodes (exact matching) and primers (allowing a 2-nucleotide mismatch). Reads containing ambiguous bases were removed. Operational taxonomic units (OTUs) were clustered with a 97% similarity cutoff using UPARSE (Edgar, 2013). The taxonomy of each 16S rRNA gene sequence was analyzed by the RDP classifier algorithm (http://rdp.cme.msu.edu/) against the Silva database (Quast et al., 2013), using a confidence threshold of 70% and implementation in QIIME (Caporaso et al., 2010). ITS sequences were processed similarly and taxonomy was assigned using the UNITE database (https://unite.ut.ee/index.php) with the RDP classifier in QIIME.