Hiseq sequencing platform

The metagenomic sequencing method was used to investigate functional changes in the gut microbiota. A total of 6 fecal samples to extract the genomic DNA (3 samples from BDB-treated BKS db mice and others from placebo-treated diabetic mice).
The qualified DNA samples were randomly interrupted with the size of about 270 bp by covaris ultrasonic crusher. The metagenomic sequencing was carried out on an Illumina HiSeq sequencing platform (Illumina Inc., San Diego, CA, USA) by Allwegene Technology Inc (Beijing, China). The raw sequence reads with quality scores below 20 accounts for more than 50% of the total read length were trimmed. We filtered the low-quality sequences of raw reads obtained by sequencing, and the subsequent analysis was based on filtered clean reads. For the host contamination of the sample, compared with the host database to filter out the reads that may come from the host. The open reading frames (ORFs) were predicted by MetaGeneMark and removed redundant by using CD-HIT software to obtain the non-redundant-nucl (https://github.com/weizhongli/cdhit). The COG functional categories were annotated by the EggNOG database via Smith-Waterman comparison algorithm. The KEGG pathway annotation was performed using a BLAST search by the KEGG database.

Genomic DNA of strain M9 was extracted using a BioTekeDNA extraction kit (Beijing, China). The 16S rRNA gene was amplified from the genome DNA via PCR using the primers 1492R and 27F [26 (link)] (Table 3). The PCR product was ligated to the pMD19-T vector (TaKaRa, kusatsu, Japen) and sequenced on an Applied Biosystems DNA sequencer (model 3730XL). The sequence of the 16S rRNA gene was compared with those in the GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 17 December 2021) and Ezbiocloud (http://www.ezbiocloud.net; accessed on 17 December 2021) databases using BLASTN. The similarity values of paired sequences were obtained by the EzBioCloud server. The genomic DNA of strain M9 was sequenced on the Illumina Hiseq sequencing platform (Majorbio, Shanghai, China). The genome assembly was performed using the ABySS 2.1.5 analysis process implemented in SMRT Link (V6.0.0.47841) to get the draft genome [48 (link)]. The genome data of strain M9 was uploaded to the Genbank database under the accession number JAEKKD000000000.1.

A total of 5 mL of rumen fluid of each sample was lysed with a shaker, and then total genomic DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen Ltd., Hilden, Germany). DNA sample concentrations were measured by a UV–VIS NanoDrop 2000c spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and DNA integrity was assessed using 1.0% agarose gel electrophoresis (Table S1 and Figure S1). Subsequently, 1.98~2.55 µg of gDNA was used for library preparation. Sequencing libraries were generated using the NEBNext^® Ultra™ DNA Library Prep Kit (NEB, Ipswich, MA, USA) following the manufacturer’s recommendations. Index codes were added to attribute sequences to each sample. Briefly, the DNA sample was fragmented by sonication to a size of 350 bp, and then the DNA fragments were end-polished, A-tailed, and ligated with a full-length adaptor for PCR amplification. Finally, the PCR products were purified (AMPure XP system, Beckman, CA, USA), and libraries were analyzed for size distribution by an (Agilent2100 Bioanalyzer, Agilent, Palo Alto, CA, USA) and quantified using real-time PCR. Metagenome sequencing was performed on an Illumina HiSeq sequencing platform (Illumina Inc., San Diego, CA, USA, 150 bp paired-end sequencing).