Miseq300 platform

Feces (0.25 g per mouse) of different cages were used in each experimental group for microbiota analysis following a previous protocol [56 (link)]. In summary, metagenomic DNA was extracted from 0.25 g of feces using the DNeasy PowerSoil Kit (Qiagen, MD, USA) using the Miseq300 platform (Illumina, San Diego, CA, USA) at the Omic Sciences and Bioinformatics Centre and Microbiome Research Unit for Probiotics in Food and Cosmetics, Chulalongkorn University. The raw sequences were quality processed and classified into operational taxonomic units (OTU) following Mothur’s standard operating platform procedures [57 (link),58 (link)]. Bioinformatic analyses included good coverage, alpha diversity (e.g., Chao), and beta diversity. Linear discriminant effect size analysis (LEfSe) and meta-stats were also performed to determine the species marker and the unique representative species of the interested group, respectively [57 (link),59 (link)].

Feces from nine mice (0.25 g per mouse) from different cages in each experimental group were divided into three samples per group (three mice per sample) before performing microbiota analysis following a previous protocol [41 (link)]. In short, metagenomic DNA was extracted from 0.25 g feces by DNeasy PowerSoil Kit (Qiagen, Germantown, MD, USA). The Universal prokaryotic 515F (forward; (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (reverse; 5′-GGACTACHVGGGTWTCTAAT-3′), with appended Illumina adapter and Golay barcode sequences, were used for 16S rRNA gene V4 library construction and sequenced using Miseq300 platform (Illumina, San Diego, CA, USA) at Omics Sciences and Bioinformatics Center, and Microbiome Research Unit for Probiotics in Food and Cosmetics, Chulalongkorn University. Raw sequences were quality processed and operational taxonomic unit (OTU) classified following Mothur’s standard operating platform procedures [42 (link),43 (link)]. Bioinformatic analyses included good’s coverage, alpha diversity (e.g., Chao), and beta diversity (e.g., non-metric multidimensional scaling (NMDS)). Linear discriminant analysis effect size (LEfSe) and meta-stats were also performed to determine species marker and unique representing species of the interested group, respectively [42 (link),44 (link)].

Mouse feces were collected by placing mice in metabolic cages (Hatteras Instruments, Cary, NC, USA) for a few hours. Then, feces (0.25 g) from each mouse in different cages were collected for microbiome analysis to avoid the influence of allocoprophagy (a habit of mice that ingest feces from other mice) on fecal microbiota analysis following previous publications [93 (link),98 (link),99 (link)]. Briefly, the metagenomic DNA was extracted from the prepared samples using a DNAeasy Kit (Qiagen, Redwood City, CA, USA). The quality and concentration of the extracted DNA were measured by nanodrop spectrophotometry. Universal prokaryotic primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with appended 50 Illumina adapter and 30 Golay barcode sequences were used for 16S rRNA gene V4 library construction in Miseq300 platform (Illumina, San Diego, CA, USA). The raw sequences and operational taxonomic unit (OTU) were classified following Mothur’s Standard Operating Procedures (SOP) [100 (link)].

PCR amplification of the 16S rRNA gene at the V3-V4 region was performed using the universal prokaryotic primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) with appended adaptor and barcodes sequences as previously reported^{26 (link),27 (link)}. Briefly, each PCR reaction was comprised of 1 × EmeraldAmp GT PCR Master Mix (TaKaRa), 0.3 μM of each primer, and 50–100 ng of metagenomic DNA in a total volume of 50 μL. The PCR conditions were 94 °C 3 min, and 25 cycles of 94 °C 45 s, 50 °C 1 min and 72 °C 1 min 30 s, followed by 72 °C 10 min. A minimum of two independent PCR reactions were performed and pooled to prevent PCR stochastic bias. Then, the 381-bp amplicon was excised after agarose gel resolution and purified using a PureDireX PCR Clean-Up & Gel Extraction Kit (Bio-Helix, Keelung, Taiwan) prior to quantification using a Qubit 3.0 Fluorometer and Qubit dsDNA HS assay kit (Invitrogen, Waltham, USA). Finally, 180 ng of each barcoded amplicon product was pooled for sequencing using the Miseq300 platform (Illumina, San Diego, CA, USA), along with the sequencing primers and index sequence^{26 (link)}. Sequencing was performed at the Omics Sciences and Bioinformatics Center, Chulalongkorn University (Bangkok, Thailand).

To obtain the genomic DNA of the microbial community, the 48 Daqu samples were extracted using an E.Z.N.A. Soil DNA Kit (Omega Bio-Tek, USA) according to the manufacturer’s instructions. The 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were applied to amplify the 16S rRNA gene of the bacterial V3-V4 variable region. The amplification procedure was as follows: 95°C predenaturation for 3 min, 27 cycles of 95°C denaturation for 30 s, 55°C annealing for 30 s, and 72°C extensions for 30 s, and 72°C extensions for 10 min. The amplification system was as follows: 4 μl 5 × FastPfu buffer, 2 μl 2.5 mmol/L dNTPs, 0.8 μl primers (5 μmol/L), 0.4 μl FastPfu polymerase, and 10 ng DNA template. The PCR products were detected on 2% (w/v) agarose gels, and the size of the paired-end sequence was no less than 550 bp. Subsequently, the PCR products were analyzed via the Illumina MiSeq 300 platform (San Diego, CA, USA).

Feces from each mouse (0.25 g per mouse; 3 mice per group) from different cages in each experimental group were collected for the microbiota analysis following a previous report [40 (link)]. Of note, mice in the same groups were housed in different cages because co-housing might induce similar gut microbiota within the same cage. In short, metagenomic DNA was extracted from individual mice by DNeasy PowerSoil Kit (Qiagen, Maryland, USA). The quality and concentration of the extracted DNA were measured by agarose gel electrophoresis and nanodrop spectrophotometry. Libraries of the V4 hypervariable region of 16S rRNA gene were amplified by polymerase chain reaction (PCR) using Universal prokaryotic primers 515F (forward; 5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (reverse; 5′-GGACTACHVGGGTWTCTAAT-3′), modified with the Illumina adapter and Golay barcode sequences in Miseq300 platform (Illumina, San Diego, CA, USA). The raw sequences and operational taxonomic unit (OTU) were classified following Mothur’s standard operating platform [41 (link), 42 (link)]. The non-metric multidimensional scaling (NMDS), the distance-based ordination method, was performed based on the Bray-Curtis dissimilarity. The 16S rDNA sequences in this study were deposited in an NCBI open access Sequence Read Archive database with accession number PRJNA776693.

According to the manufacturer’s instructions, the microbial genomic DNA was extracted from cecum contents of yellow-feathered broilers using the QIAamp DNA Stool Mini Kit (QIAGEN, US). The concentration of DNA in the extracted samples was detected with a NanoDrop 2000 spectrophotometer (ThermoFisher, US). The extracted DNA samples above were submitted to Shanghai Majorbio Bio-pharm Technology Co, Ltd for sequence analysis. The V3–V4 region of the bacterial 16S rRNA gene was amplified from each genomic DNA sample by using the primers 338F (5′-ACT CCT ACG GGA GCA GCA-3′)—806R (5′-GGA CTA CHV GGG TWT CTA ATT-3′). Sequencing libraries were then constructed using TruSeqTM DNA Sample Prep Kit and sequenced on an Illumina MiSeq 300 platform. After sample splitting of PE reads obtained by MiSeq sequencing, double-ended reads were firstly controlled and filtered according to sequencing quality and, at the same time, spliced according to the overlapping relationship between double-ended reads to obtain optimized data after quality control spliced. Then, sequence denoising methods (DADA2/Deblur) were used to process the optimized data, and Amplicon Sequence Variant (ASV) was used to represent the sequence and abundance information.