title: Lactobacillus plantarum ZDY04 exhibits strain-specific property of lowering-TMAO via modulation of gut microbiota in mice
design: Precipitate fractions of cecal contents were used to extract metagenomic DNA through the cetyltrimethylammonium bromide method (Schlüter et al. 2008). Subsequently, gut microbiota composition was analyzed by amplifying the V4 domain of bacterial 16S ribosomal DNA (16S V4). The 16S V4 was amplified using a barcoded primer pair (515F: 5′-GTGCCAGCMGCCGCGGTAA-3′; 806R: 5′-GGACTACHVGGGTWTCTAAT-3′) (Caporaso et al. 2011). PCR was performed in 30 μL reactions containing 15 μL of Phusion® High−Fidelity PCR Master Mix (New England Biolabs, Ipswich, MA, USA), 0.2 μM each of forward and reverse primers, and approximately 10 ng of template DNA. Thermal cycling was performed under the following conditions: initial denaturation at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 50 °C for 30 s, elongation at 72 °C for 30 s, and final extension at 72 °C for 5 min. PCR products with lengths between 400 and 450 bp were chosen and purified using GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Carlsbad, CA, USA). Sequencing libraries were generated using NEB Next® Ultra™ DNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA) following the manufacturer's instructions, and index codes were added. Library quality was assessed on a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Carlsbad, CA, USA) and Agilent Bioanalyzer 2100 system. Finally, the library was sequenced on an Illumina HiSeq platform, generating 250−bp paired-end reads. Paired-end reads from the original DNA fragments were merged by FLASH and assigned to each sample by unique barcodes. Sequences with ≥97% similarity were assigned to the same operational taxonomic units (OTUs). A representative sequence was selected for each OTU, and the RDP classifier was used to annotate taxonomic information for each representative sequence. Sequences were analyzed using quantitative insights into microbial ecology and R software (Version 2.15.3) to analyze alpha (within sample) and beta (among sample) diversities.