Quantifluor st fluorescence quantitative system

Microbial DNA was extracted using the E.Z.N.A.® DNA Kit (Omega Bio-tek, Norcross, GA, US) according to the manufacturer’s protocols. The V4-V5 region of the bacterial 16S rRNA gene was amplified by Polymerase Chain Reaction (PCR) using the forward primer 515F (5′-barcode-GTGCCAGCMGCCGCGG-3′) and reverse primer 907R (5′-CCGTCAATTCMTTTRAGTTT-3′), where the barcode was an eight-base sequence unique to each sample. The following cycling parameters were used: 95 °C for 2 min, followed by 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s and a final extension at 72 °C for 5 min. Amplicons were extracted from 2% agarose gels and purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, US), according to the manufacturer’s instructions, and quantified using the QuantiFluor™-ST fluorescence quantitative system (Promega, Madison, WI, US). Equimolar amounts of the purified amplicons were then pooled and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard protocols.

The bacterial communities in the fecal samples were investigated by Illumina MiSeq high-throughput sequencing. The V3 and V4 regions of the 16S rDNA gene were selected for PCR. The primers were barcoded as 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′), where the barcode was an eight-base sequence unique to each sample. The 20-μl PCR reaction mixture was composed of 4 μl of 5 × FastPfu buffer, 2 μl of 2.5 mM dNTPs, 5 μM each of forward and reverse primer, 0.4 μl TransStart Fastpfu DNA Polymerase (TransGen Biotech, Beijing, China), and 10 ng DNA template. The following cycling parameters were used: maintenance at 95°C for 2 min, 27 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 5 min. Triplicate reaction mixtures were pooled for each sample, purified using an AxyPrep DNA gel extraction kit (Axygen, Union City, CA, United States), and quantified using a QuantiFluor-ST fluorescence quantitative system (Promega, Madison, WI, United States). Amplicons from different samples were sent out for sequencing on an Illumina MiSeq platform at Shanghai Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

The gut digesta was immersed in liquid nitrogen for 10 min and then freeze-dried to remove the residual ethanol. DNA from each sample was extracted with a ZR Soil Microbe DNA MiniPrep kit, according to the manufacturer’s instructions. After quality and purity evaluation, the V3–V4 region of the 16S rRNA was amplified from the extracted DNA using the bacteria-specific primer pair 338F (forward: 5′-ACTCCTACGGGAGGCAGCAG-3′) (Mori et al., 2014 (link); Xu et al., 2016 (link)) and 806R (reverse: 5′-GGACTACHVGGGTWTCTAAT-3′) and the GeneAmp^®9700 thermal cycler (Applied Biosystems, Foster City, CA, United States) (Sun et al., 2014 (link)). The fragments (421–460 bp in length) were purified using the AxyPrep DNA gel extraction kit (Axygen Bio-sciences, Union City, CA, United States) and QuantiFluor^TM-ST fluorescence quantitative system (Promega, Madison, WI, United States). The purified amplicons were mixed at equimolar concentrations and then sequenced following a paired-end 250 bp × 2 strategy on an Illumina MiSeq platform operated by Majorbio Bio-Pharm Technology Co., Ltd., (Shanghai, China). The original reads were uploaded to the NCBI Sequence Read Archive database (Accession Number: PRJNA492364).

Prior to sequencing, DNA quality was analyzed on an Agilent 2100 Bioanalyzer (Agilent, United States), and concentrations were determined using a QuantiFluor^TM-ST Fluorescence Quantitative System (Promega Company, United States). After quantification, DNA was mixed in equimolar ratios according to the sequencing requirements. Paired end (PE) libraries were constructed by PCR using the TruSeq^TM DNA Sample Prep Kit (Illumina). Illumina MiSeq sequencing was performed at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

Small pieces of coral sample including tissue, mucus and skeleton (approximately 50 mg), cut with a pair of scissors, were used to extract genomic DNA using TIANamp Marine Animals DNA Kit [Tiangen Biotech (Beijing) Co., Ltd., Beijing, China] according to the manufacturer’s instructions. The V3–V4 region of bacterial 16S rRNA gene was amplified using the bacterial-specific forward primer 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and reverse primer 806R (5′-GGACTACHVGGGTWTCTAAT-3′), where barcode is an eight-base sequence unique to each sample (Mori et al., 2014 (link); Xu et al., 2016 (link)). The reaction system and procedure of PCR using a ABI GeneAmp^® 9700 thermal cycler are the same as previously described (Sun et al., 2014 (link)). Triplicate PCR products were pooled for each sample, and then fragments with size in the range of 421–460 bp were purified and quantified using the AxyPrep DNA gel extraction kit (Axygen Biosciences, Union City, CA, United States) and QuantiFluor^TM-ST Fluorescence quantitative system (Promega, United States). Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 250) on an Illumina MiSeq platform according to the standard protocols (Majorbio Bio-Pharm Technology Co. Ltd., Shanghai, China). The raw reads were deposited into the NCBI Sequence Read Archive (SRA) database (Submission Number: SUB2295385).