Quantifluor fluorometer

DNA from the fecal material was extracted using a QIAamp DNA Stool Mini Kit (QIAGEN, Dusseldorf, Germany) following the standard protocol. DNA concentration was determined using a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA). The V3 and V4 regions of 16S rDNA from all samples were amplified using the 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) primers. The polymerase chain reaction (PCR) products were quantified and purified using a QuantiFluor™ fluorometer (Promega Biotech, Madison, WI, USA). Negative controls included no templates for DNA extraction and PCR amplification. PCR products were mixed in equidensity ratios. The mixture of PCR products was purified with a Gel Extraction Kit (QIAGEN). Sequencing libraries were generated using the TruSeq^® DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, CA, USA) following manufacturer’s instructions and index codes were added. The library quality was assessed on the Qubit@ 2.0 Fluorometer (Thermo Fisher Scientific) and the Bioanalyzer 2100 system (Agilent, Santa Clara, CA, USA). Finally, the library was sequenced on a HiSeq2500 platform (Illumina) and 250 bp paired-end reads were generated.

The V3–V4 region of 16s rRNA gene was amplified by PCR using universal primers 341F: CCTACGGGNGGCWGCAG and 806R: GGACTACHVGGGTATCTAAT (Guo et al., 2017 (link)). PCR amplification was performed using high-fidelity KOD polymerase (NEB, Ipswich, UK). PCR reactions were performed in triplicate using 50-μL mixtures containing 5 μL of 10× KOD buffer, 5 μL of 2 mM dNTPs, 3 μL of 25 mM MgSO₄, 1.5 μL of each primer (10 μM), 1 μL of KOD polymerase, and 100 ng of template DNA. PCR reagents were obtained from TOYOBO, Japan. PCR conditions were 94 °C for 2 min, followed by 30 cycles at 98 °C for 10 s, 62 °C for 30 s, and 68 °C for 30 s and a final extension at 68 °C for 5 min. The amplicons were pooled, purified, and then quantified using the QuantiFluor™ fluorometer (Promega, Beijing, China). Finally, the amplicons were sequenced using the paired-end strategy (PE250) on the Illumina HiSeq 2500 platform, following standard protocols. The raw reads were deposited into the NCBI Sequence Read Archive database (PRJNA778015).

Total genomic DNA was extracted from fecal samples using a DNA stool kit according to the instructions. The V3–V4 hypervariable region of the 16S rRNA sequence was amplified by PCR with specific primers 338F (5′-ACTCCTACGGAGGCAGCAGCAG-3′) and 806R (5′-GGACTACHVGGTWTCTAAT-3′). After being quantified with QuantiFluor™ fluorometer (Promega, Milano, Italy), purified PCR amplicons were mixed in equal amounts, and connected through sequencing joints. Finally, a sequencing library was constructed, and the sequencing was performed on Illumina HiSeq PE250 (Illumina, CA, United States). The amplicon sequencing data were submitted to the NCBI SRA (SubmissionID, SUB11291689; BioProject ID, PRJNA823644).

DNA was then extracted using the HiPure Stool DNA Kit (Magen, Guangzhou, China), and their quantity and quality were determined using the Qubit^® 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and by agarose gel electrophoresis. Subsequently, the V4 region of the bacterial 16S rRNA gene was amplified in 50 μL reaction mixtures using 2× Taq master Mix (TransGen Biotech Co., Ltd., Beijing, China) with 5 μM each of the primers 515F and 806R.
According to the operation manual (Gene Denovo Biotechnology Co., Ltd., Guangzhou, China), the thermal cycling conditions were as follows: initial denaturation at 95 °C for 2 min followed by 27 cycles at 98 °C for 10 s, 62 °C for 30 s, and 62 °C for 30 s, and a final extension at 68 °C for 10 min. The products were recovered and quantified with a QuantiFluor™ fluorometer (Promega, Madison, WI, USA). The PCR amplicons were sequenced on the Illumina HiSeq2500 platform (Novogene Bioinformatics Technology Co. Ltd., Tianjin, China). The raw data were deposited in the Sequence Read Archive database of NCBI (BioProject: PRJNA766330).

Pooled amplicons were purified using the E.Z.N.A^® Cycle Pure Kit (OMEGA, Bio-tek) and Cycle-Pure Spin Protocol according to the manufacturer’s instructions. Sequencing was conducted at the Lund University Sequencing Facility, Sweden. Pooled amplicons were reduced for short fragments by using Agencourt AMPure XP (Beckman Coulter) and inspected using a DNA 1000 kit on a 2100 Bioanalyzer (Agilent). Amplicons were quantified using the Quant-iT dsDNA assay kit (Invitrogen) and Quantifluor fluorometer (Promega), and pools were diluted to obtain a total of 1×10⁷ copies μL⁻¹. Titration and library production (aiming at 10–15% enrichment) were performed using emulsion PCR and the Lib-A kit (Roche). DNA-positive beads were enriched, counted on an Innovatis CASY particle counter (Roche), processed using the XLR70 sequencing kit (Roche), and loaded onto a picotiter plate for pyrosequencing on a 454 Life Sciences Genome Sequencer FLX machine (Roche). DNA sequences were archived at NCBI SRA under the accession number SRP039011.

For metagenomic sequencing, total DNA from each kefir beverage was extracted using Meta-G-Nome DNA Isolation Kit (Epicentre) according to the manufacturer’s instructions. DNA integrity was confirmed by agarose electrophoresis, and DNA concentration was determined by UV absorbance using a QuantiFluor^® fluorometer (Promega, Madison, WI, USA).