Hiseq pe250

Stool samples were collected into sterile containers 12 h after the last gavage and immediately frozen at −80°C. Microbial DNA was extracted from the fecal material of each sample using the QIAamp DNA Stool Minikit (Qiagen Ltd, Strasse, Germany). The next-generation sequencing of 16S rDNA using the Illumina HiSeq PE250 was performed by Realbio Genomics Institute (Shanghai, China). V3–V4 region of the 16S rDNA gene sequence were amplified from genomic DNA using the primer pair, F341 (5′-ACTCCTACGGGRSGCAGCAG-3′) and R806 (5′- GGACTACVVGGGTATCTAATC-3′). The raw data were then subjected to a quality control procedure using UPARSE. Cluster reads into operational taxonomic units (OTUs) of ≥97% similarity using Usearch. Principal coordinate analysis (PCoA), heatmap analysis, and species abundance analysis were performed using R. Relative abundance of gut microbiota was compared between the control, PG, and rifaximin groups using linear discriminant analysis effect size (LEfSe) coupled with the Kruskal-Wallis rank sum test. Thresholds for the LEfSe analysis was log linear discriminant analysis (LDA) score >2. PICRUS was used to predict metagenome function from the 16S rRNA data (Qi et al., 2018 (link)). The 16S rRNA gene sequences have been deposited into the NCBI Sequence Read Archive (SRA) database under accession number SRP182773.

Extraction and purification of microbial DNA from the distal ileal contents of rats were performed as instructed in the QIAamp Fast DNA Stool Mini Kit (Qiagen). DNA was then submitted to the PCR amplification of the 16S V3-V4 region of bacteria. Primer sequences are as follows: forward primer (5′-3′) ACTCCTACGGGRSGCAGCAG and reverse primer (3′-5′) GGACTACVVGGGTATCTAATC. The KAPA HiFi HotStart Ready Mix PCR kit was used to ensure the accuracy and efficiency of the PCR amplification. DNA library should have a quality inspection and quantitative analysis with Qubit. The 16S rDNA sequencing was performed and analyzed with the Illumina HiSeq PE250in Realbio Genomics Institute (Shanghai, China).

Bacterial genomic DNA was extracted from frozen sow fecal samples and neonatal piglet colonic contents with an E.Z.N.A. TM Stool DNA kit (Omega Bio-Tek, Norcross, GA, USA). Sequencing and data analysis were subsequently performed on the Illumina HiSeq PE250 platform by Novogene (Beijing, China), as previously described in Zhou et al. [73 (link)]. Sequences were clustered into the same OTUs at 97% sequence similarity. The alpha diversity and beta diversity were calculated for comparison of taxonomic data. The observed species, Chao 1 index, and Shannon index were used to determine differences in alpha diversity according to different diets. Unifrac weighted distance matrices were calculated, and analysis of similarities (Anosim) was used to access differences among the microbial communities. All analyses from clustering to alpha and beta diversity were performed in QIIME (V1.7.0) and displayed in R software (V2.15.3).

Feces from mice were collected on day 14 after fluconazole treatment and were immediately stored at-80°C. The fecal samples were mashed in PBS containing 0.5% Tween20 solution and were then subjected to repeated freezing at-80°C for 10 min and thawing at 60°C for 5 min. The next steps involved extracting and purifying the DNA using the phenol-chloroform isoamyl alcohol method and a commercial reagent (Solarbio, Beijing, China). Next, the concentration and purity of total DNA were determined by using a NanoDrop spectrophotometer (Thermo Scientific, MA, United States). The hypervariable region 4 (V4) of the bacterial 16S rRNA was amplified by a polymerase chain reaction (PCR) using primers V4-F (5′-GTGTGYCAGCMGCCGCGGTAA-3′) and V4-R (5′-CCG GACTACNVGGGTWTCTAAT-3′). DNA sequencing was performed on an Illumina Hiseq PE250, and the raw data of 16S rRNA gene sequencing were analyzed with the QIIME2 platform (v2020.2).

The Mo Bio PowerFecal^TM DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) was utilized to extract the microbial DNA of thawed fecal sample (0.5 g). A nucleic acid/protein analyzer (Beckman DU-800, Beckman Coulter, Inc., CA, USA) was used to detect the concentration and purity of the genomic DNA. Following this, DNA samples were sent to perform amplicon pyrosequencing on Illumina HiSeq PE250 platforms at Novogene Bioinformatics Technology in Beijing, China. The V4 hypervariable region of the 16S rRNA gene was amplified using a forward primer 515f (5′-GTGCCAGCMGCCGCGGTAA-3′) and a reverse primer 806r (5′-GGACTACHVGGGTWTCTAAT-3′).
Operational taxonomic units (OTUs) come from high-quality tags utilizing Uparse v7.0.1001 (http://drive5.com/uparse/) at an identity threshold of 97% used Ribosomal Database Project (RDP) classifier Version 2.2 (http://github.com/rdpstaff/) to assign taxonomy for 16S rRNA gene sequences. The relative abundance of each OTU was explored at phylum and genus levels. A Venn diagram was generated for a comparison of the OTUs of the two treatments. Alpha diversity values for each sample were assessed by Qiime 1.7.0. (http://qiime.org/home_static/dataFiles.html) Principal coordinates analysis (PCoA) plots were produced using unweighted UniFrac metrics.

After DNA extraction, the V4 region of the 16S rRNA gene was amplified using specific barcode primers (V4F, 5′‐GTGTGYCAGCMGCCGCG GTAA‐3′, and V4R, 5′‐CCGGACTACNVGGG TWTCTAAT‐3′). The PCR amplification products were mixed in equal amounts measured by QuantiFluor. All samples were subjected to paired‐end sequencing on the Illumina Hiseq PE250 (San Diego, CA, USA) platform. High‐throughput sequencing analysis of bacterial rRNA genes was processed using the Quantitative Insights into Microbial Ecology (QIIME, version 1.9.1) software suite. The calculated p‐value was gone through FDR Correction, taking FDR ≤0.05 as a threshold (Deng et al., 2021 ).

High-throughput sequencing was performed with the Illumina HiSeq PE250 platform at the Shanghai Ruiyi Biotechnology Co. Ltd. (Shanghai, China). Amplification of the highly variable V3-V4 region of the 16S rRNA gene was conducted using universal primers 341F (5′-CCTACGGGRSGCAGCAG-3′) and 806R (5′-GGACTACVSGGGTATCTAAT-3′). Raw paired-end reads were assembled by PANDAseq, and the following conditions were used to define the quality filter: reads with an average quality score of more than 20, reads with less than three ambiguous N bases, and reads with a long range from 220 to 500 nucleotides [26 (link)]. After the singletons were discarded, the sequences were clustered into operational taxonomic units (OTUs) using the software USEARCH at ≥97% similarity [27 (link)]. Linear discriminant analysis (LDA) effect size (LEfSe) was conducted to assess the effect size of each differentially abundant OUT [28 (link)]. Subsequent bioinformatics steps were analyzed using a QIIME software package [29 (link)].