Sequencing of the 16S rRNA gene

DNA was extracted from cell pellets of the enrichment culture using the DNeasy PowerSoil Kit (Qiagen Japan, Tokyo, Japan) according to the manufacturer’s protocols. DNA concentrations were assessed using Qubit dsDNA BR assay kits and a Qubit 3.0 fluorospectrometer (Thermo Fisher Scientific, Waltham, MA, USA). Extracted DNA was processed as follows for direct and amplicon sequencing of the 16S rRNA gene. In direct sequencing, the PCR amplification of a partial 16S rRNA gene sequence and Sanger sequencing were performed as previously described (39) with slight modifications. PCR amplification used the oligonucleotide primers 515F (5′-GTGCCAGCMGCCGCGGTA-3′)–806R (5′-GGACTACHVGGGTWTCTAAT-3′) (8) for a prokaryotic 16S rRNA gene. The PCR mixture had a volume of 100 μL and contained 4–100 ng of DNA, oligonucleotide primers (0.3 μM each), dNTPs (200 μM), 1× PCR buffer, and PrimeSTAR GXL Taq (0.025 U μL⁻¹) (Takara Bio, Kusatsu, Japan). Cycling conditions were as follows: 40 cycles at 98°C for 10 s, followed by 50°C for 15 s, then 72°C for 45 s; and finally 72°C for 5 min. PCR products were purified using FastGene Gel/PCR Extraction Kits (Nippon Genetics, Tokyo, Japan). The direct sequencing of the purified PCR amplicon was performed using the 515F and 806R primers as sequencing primers. The nucleotide sequences identified were assembled manually and subjected to a blastn search using the nr database as a reference (National Center for Biotechnology Information, NCBI).

Amplicon sequencing of the 16S rRNA gene was performed as previously described (19). PCR amplification used the above-described oligonucleotide primers 515F and 806R containing Illumina tag sequences at the 5′ ends. The amplicon was purified and used in the preparation of a library by means of MiSeq Reagent Kits v2 nano (Illumina, San Diego, CA, USA) for sequencing on Illumina MiSeq (Illumina). Amplicon library concentrations were measured using BioAnalyzer DNA 1000 (Agilent Technologies, Santa Clara, CA, USA). The quality of the sequencing analysis was verified by examining the PhiX library prepared from the PhiX spike-in control (Illumina). Sequence reads with a low quality score (Phred quality score ≤30) were eliminated using the fastx trimmer tool, and paired-end sequence reads were then assembled in the paired-end assembler for the Illumina sequence software package (PANDAseq) (33). Sequence reads with ≥97% similarity were grouped into an operational taxonomic unit (OTU) by the UCLUST algorithm (14). The phylogenetic affiliations of the OTUs were identified using a blastn search against reference sequences in the Greengenes database version 13_5 (13) and in the nr database provided by the NCBI.