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.