Pe150 sequencing

DNA was extracted from the pure culture using a Bacterial Genome DNA Rapid Extraction Kit (Huiling Biotechnology Co., Ltd., Shanghai, China), following the manufacturer’s instructions. The exact concentration of the DNA was measured using a Qubit 4 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA), and DNA purity and integrity were detected via agarose gel electrophoresis. Genome sequencing was completed using PacBio sequencing technology and Illumina PE150 sequencing for high-accuracy assembly. The genome was assembled using SMRT Link software (version 5.0.1) [31 (link)], and the protein-coding genes were predicted using GeneMarkS software (Version 4.17) [32 (link)]. The tRNA was predicted by the software tRNAscan-SE (version 1.3.1) [33 (link)], the rRNA genes were predicted using RNAmmer software (version 1.2) [34 (link)], and small RNA was identified against the Rfam database [35 (link)] using CMsearch (version 1.1rc4) [36 (link)]. The genomic island was predicted using IslandPath-DIOMB (version 0.2) [37 (link)], and the CRISPR (clustered regularly interspaced short palindromic repeat) sequences were predicted using CRISPRdigger software (version 1.0) [38 (link)]. A display of the entire genomic map was generated using Circos (version 0.69) [39 (link)], along with the prediction results of the protein-coding genes.

Fecal samples were collected from all experimental mice beginning in the last week of 14 weeks. The collected fecal pellets were immediately transferred into liquid nitrogen and then stored at −80°C. Subsequently, the DNA was extracted using a FastDNA^M SPIN kit for feces (catalogue no. 116570200, MP Biomedicals SARL, Illkirch, France) and processed according to the manufacturer’s instructions. The extracted DNA was sonicated and randomly broken into fragments with lengths of approximately 300 to 350 bp, and then the base A and an adapter were added to the 3′ end of the DNA fragment to repair the modified DNA fragment. This was followed by purification and PCR amplification to complete the library preparation. Qubit 2.0 was used for preliminary quantitative analysis; the library was diluted to 2 ng/μL, and the insert fragment size of the library was detected using Agilent 2100. After reaching the preset values, quantitative PCR (qPCR) was performed to accurately quantify the effective concentration of the library to ensure optimum quality. Illumina PE150 sequencing was conducted after pooling different libraries according to the effective concentration and the target data volume (Novogene, China).

DNA concentrations were determined using the Qubit dsDNA assay kit after DNA extraction from fecal samples, with OD values between 1.8 and 2.0 and DNA content above 1 μg to construct libraries. DNA was randomly interrupted into fragments of approximately 350 bp in length using a Covaris ultrasonic disruption instrument, and PCR products were purified by end repair, A‐tail addition, and sequencing adapters (AMPure XP system). Initial quantitation was performed using Qubit 2.0, diluting the library to 2 ng/μl, followed by detection of inserts into the library using Agilent 2100, and accurate quantitation of the effective concentration of the library (effective concentration of the library >3 nM) was performed using Q‐PCR method after inserts were as expected. The index‐encoded samples were clustered on a cBot cluster generation system. After cluster generation, Illumina PE150 sequencing was performed and paired‐end reads were generated.