Metavx library preparation kit

One milliliter of the saliva sample was thawed at room temperature and homogenized by vortex. QIAamp^® DNA Minikit (Qiagen, Valencia, CA, USA) was employed for DNA extraction. Extracted DNA samples were sent to Vishuo Biomedical in Singapore to perform 16S rDNA sequencing by Illumina Miseq. In short, a MetaVx™ Library Preparation kit (Genewiz, South Plainfield, NJ, USA) was used in library preparation. A V3–V4 region of rDNA was selected as a target. The amplicons were generated using forward primers containing the sequence 5′-CCTACGGRRBGCASCAGKVRVGAAT-3′ and reverse primers containing the sequence 5′-GGACTACNVGGGTWTCTAATCC-3′. DNA libraries were multiplexed and loaded on Illumina MiSeq according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). Sequencing was performed using a 2 × 300 base pair. Raw 16s rRNA sequencing data are available with bioproject accession number PRJNA736207.

DNA was extracted from water filter membranes and dried sediment using a modified hexadecyltrimethylammonium bromide (CTAB) method [42] (link), [43] (link), [44] . All extracted metagenomic DNA was dissolved in TE buffer and stored at −20 °C until further use.
Metagenomic DNA were quantified by using a Qubit® 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and the quality of DNA was assessed on 0.8% agarose gels. Approximately 5–50 ng of DNA was used as template for amplifying the V4–V5 hypervariable region of the 16S rRNA gene of microbiota for each sample. Sequences for the paired primers are “GTGYCAGCMGCCGCGGTAA” and “CTTGTGCGGKCCCCCGYCAATTC”, respectively [24] (link). The sequencing library was constructed using a MetaVx™ Library Preparation kit (GENEWIZ, Inc., South Plainfield, and NJ). The ends of the 16S rDNA amplicons were added with indexed adapters by limited cycle PCR. Sequencing libraries were verified using the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA) and quantified by Qubit® 2.0 and quantitative PCR (Applied Biosystems, Carlsbad, CA). All amplicons were sequenced on the Illumina MiSeq platform (paired-end, 2 * 300 bp). All sequencing data for the 14 water samples and the 14 sediment samples were deposited into NCBI’s Sequence Read Archive (SRA) database under the Bioproject number PRJNA352457.

The aseptic operation was performed strictly to collect intestinal content which immediately froze at −80 ℃ for subsequent experiments. 16S rDNA amplicon sequencing and Illumina MiSeq were conducted at GENEWIZ. In brief, sample genomic DNA was extracted and qualified for the amplification and sequencing library construction of the 16S rDNA variable region by using a MetaVx Library Preparation kit (GENEWIZ) and then implementing high-throughput sequencing [32 (link)]. Bioinformation analysis first filters the original data, obtains effective sequences and then conducts cluster analysis to acquire operational taxonomic units (OTU) of different species to obtain species distribution information of each sample. Based on OTU analysis results, species richness and evenness of each group gained by multiple α diversity index analysis and depended on taxonomic information, community structure statistical analysis carried out at various taxonomic levels. The differences in community structure between different groups are intuitively displayed via the unweighted pair group method with arithmetic mean (UPGMA) cluster tree, principal co-ordinates analysis (PCoA), principal co-ordinates analysis (PCA), and so on.