Miseq 2 250 platform

DNA from oral biofilm and gut samples of six animals per group was extracted using the Master pureTM DNA Purification Kit (Epicentre^® Illumina Company, Madison, WI, USA). A barcoded primer set Bakt_341F CCTACGGGNGGCWGCAG and Bakt_805R GACTACHVGGGTATCTAATCC [25 (link)] was used to amplify the hypervariable V3–V4 region of 16SrRNA. DNA was sequenced by ByMyCell (Ribeirão Preto, São Paulo, Brazil) using the Illumina MiSeq 2 × 250 platform. Data were submitted to Sequence Read Archive (SRA) under BioProject identification #PRJNA994574.

All samples subjected to DNA extraction were obtained from inside the feces using sterile tweezers to avoid soil contamination with an equal weight of 75 mg. Total genomic DNA was extracted from fecal samples using the E.Z.N.A^® Stool DNA Kit (Omega Biotechnology, USA) according to manufacturer’s instruction, and was stored at − 70 °C before further analysis. DNA was amplified by using the 515f/806r primer set (515f: 5′-GTG CCAGCMGCCGCGGTA A-3′, 806r: 5′-XXX XXXGGACTACHV GGGTWT CTA AT-3′), which targets the V4 region of the microbial 16S rRNA, with the reverse primer containing a 6-bp error-correcting barcode unique to each sample. PCR reaction was performed using phusion high-fidelity PCR Mastermix (New England Biolabs LTD., Beijing, China) with the following condition: 94 °C for 3 min (1 cycle), 94 °C for 45 s/50 °C for 60 s/72 °C for 90 s (35 cycles), and a last step of 72 °C for 10 min. PCR products were purified by using the QIAquick Gel Extraction Kit (QIAGEN, Dusseldorf, Germany). Sequencing was conducted on an Illumina MiSeq 2 × 250 platform according to protocols described by Kozich et al. (2013 (link)). Sequencing of partial 16S RNA genes was performed by the Novogene Bioinformatics Technology Co., Ltd. (Beijing, China).

Illumina MiSeq sequencing was performed at the Chengdu Institute of Biology, Chinese Academy of Sciences. Briefly, DNA samples were amplified using the primer set 515F/806R (515F: 5′-GTGCCAGCMGCCGCGGTAA-3′, 806R: 5′-AACGCACGCTAGCCGGACTACVSGGGTATCTAAT-3′), which targets the V4 region of the bacterial 16S rDNA. The polymerase chain reaction (PCR; total volume, 100 μL) comprised 0.75 units of Ex Taq^® DNA polymerase (Takara, Dalian, China), 1× Ex Taq^® loading buffer (Takara), 0.2 mM dNTP mix (Takara), 0.2 μM of each primer, and 100 ng of template DNA. Thirty-five PCR cycles were performed (95°C for 3 min, 94°C for 100 s, 50°C for 1 min, 72°C for 1 min, and 72°C for 10 min). Finally, as described by Caporaso et al. [25 (link)], pyrosequencing was performed on an Illumina MiSeq 2 × 250 platform.

The specific primers 515F and 806R were used for the sequencing of the V4 hypervariable region, as suggested by the Earth Microbiome Project [21 (link)] and as previously described [22 (link)]. DNA libraries was sequenced at the Sequencing Unit in the National Institute of Genomic Medicine (INMEGEN) by an Illumina Miseq 2 × 250 platform (Illumina, San Diego, CA, USA) [23 (link)].
Processing of the Illumina fastq reads was performed using the QIIME 1.8.(16). The UCHIME algorithm was used for detection and removal of chimeric sequences. The Greengenes database was used as the reference 16S database. Taxonomy was assigned by the Ribosomal Database Project (RDP) classifier using a threshold of 80% [24 (link)]. The taxonomic composition of the gut microbiota was analyzed by METAGENassist.

DNA was amplified using the 515f/806r primer set (515f: 5′-GTG CCA GCM GCC GCG GTA A-3′, 806r: 5′-XXX XXX GGA CTA CHV GGG TWT CTA AT-3′), which targets the V4 region of the bacterial 16S rDNA. The reverse primer contains a 6-bp error-correcting barcode unique to each sample. PCR amplifications were performed in a 30-μl mixture containing 15 μl of Phusion High-Fidelity PCR Master Mix (New England Biolabs, UK), 0.2 μM forward and reverse primers, 10 ng of template DNA and nuclease-free water up to 30 μl. The PCR conditions were 98 °C for 1 min (1 cycle), then 98 °C for 10 s, 50 °C for 30 s and 72 °C for 60 s (30 cycles), followed by 72 °C for 5 min. The PCR products were verified by 2% agarose gel electrophoresis and mixed in equidense ratios. The mixture of PCR products was purified using a GeneJET Gel Extraction Kit (Thermo Scientific, USA). Sequencing libraries were generated using a NEB Next Ultra DNA Library Prep Kit for Illumina (New England Biolabs, UK). Sequencing was conducted on an Illumina MiSeq 2 × 250 platform at BGI, Inc. (Shenzhen, China) according to protocols described by Caporaso et al. (2012 (link)) and Kozich et al. (2013 (link)).

Total bacteria DNA was extracted from fecal samples by using PowerFecal™ DNA Isolation kit (MO BIO Laboratories, Carlsbad, CA, USA) according to manufacturer's instruction, and was stored at −80°C before further analysis. Sequencing was performed at the Novogene Bioinformatics Technology Co., Ltd. Briefly, DNA was amplified by using the 515f/806r primer set (515f: 5′-GTG CCA GCM GCC GCG GTA A-3′, 806r: 5′-XXX XXX GGA CTA CHV GGG TWT CTA AT-3′), which targets the V4 region of the bacterial 16S rDNA, with the reverse primer containing a 6-bp error-correcting barcode unique to each sample. PCR reaction was performed using phusion high-fidelity PCR Mastermix (New England Biolabs (Beijing) LTD., China) with the following condition: 94°C for 3 min (1 cycle), 94°C for 45 s/50°C for 60 s/72°C for 90 s (35 cycles), and a last step of 72°C for 10 min. PCR products were purified by using the QIAquick Gel Extraction Kit (QIAGEN, Dusseldorf, Germany). Pyrosequencing was conducted on an Illumina MiSeq 2 × 250 platform according to protocols described by Caporaso, et al12 (link).