title: DNA-Seq of Bifidobacterium breve
design: 1.Objective: The primary goal of this study is to elucidate the molecular mechanisms underlying propionate production in the probiotic strains Bifidobacterium breve XA-2103 and Pediococcus acidilactici GOLDGUT-PA0755. These strains, derived from human fecal samples, are of particular interest due to their potential role in modulating gut health through short-chain fatty acid (SCFA) production.2.Experimental Design: We have extracted the strains Bifidobacterium breve XA-2103 and Pediococcus acidilactici GOLDGUT-PA0755 from human fecal samples and plan to sequence their genomes individually using next-generation sequencing (NGS) technology. The sequencing approach will involve single-strain genomic sequencing to gain insights into the genetic basis of propionate production.3.Sample Information: Detailed information regarding the sample collection, including the location, time, conditions, and the health status of the donors, has been meticulously documented. The samples were collected from healthy human subjects, ensuring the relevance of the strains to human gut microbiota.4.Sequencing Strategy: The sequencing will be performed using the Illumina platform, employing paired-end sequencing to generate high-quality genomic data. The genomic DNA from the samples will be extracted using the STE method, ensuring high purity and integrity. The quality will be assessed using agarose gel electrophoresis, and the quantity will be determined using a Qubit fluorometer.5.Library Preparation: The qualified DNA samples, confirmed by electrophoresis, will be fragmented to approximately 350bp using the Covaris ultrasound disruptor. The NEBNextUltra DNA Library Prep Kit for Illumina (NEB, USA) will be utilized for end-repair, A-tailing, adapter ligation, purification, and PCR amplification to complete the library preparation process. Following the construction, the library will be quantified using Qubit 2.0 and diluted to a concentration of 2ng/ul. The insert size will be verified using the Agilent 2100, and the effective concentration will be accurately quantified using qPCR to ensure library quality.6.Scientific Inquiry: The study aims to investigate whether the administration of these propionate-producing probiotics to mice with colitis can alter intestinal barrier integrity and ameliorate symptoms of the disease.7.Research Background: There is a positive correlation between the levels of SCFAs in the gastrointestinal tract and the diversity of the gut microbiota. In turn, a higher diversity of gut microbiota is associated with enhanced intestinal barrier integrity. Acetic acid, butyric acid, and propionic acid are the primary SCFAs in the human gastrointestinal tract. While the beneficial effects of butyric acid on gastrointestinal health have been established, our study focuses on propionic acid, as it is another key SCFA with potential health benefits. The discovery of probiotics that produce high levels of these SCFAs could have significant implications for gastrointestinal health.