Mgieasy circularization kit

RNeasy Mini Kit (Qiagen, Venlo, Netherlands) was used to extract total RNA from the bacterial samples. The total RNA samples were submitted to Bioengineering Lab (Sagamihara, Japan). After removal of ribosomal RNA using riboPOOLS (siTOOLs Biotech, Planegg, Germany), a cDNA library for RNA-seq analysis was generated using MGIEasy RNA Directional Library Prep Set (MGI Tech, Shenzhen, China). The cDNA library was used to construct a circular DNA library using the MGIEasy Circularization Kit (MGI Tech). The cDNA library anchored by DNA Nanoball (DNA) was subjected to sequencing analysis using DNBSEC-G400 (MGI Tech). Nucleic Acid SeQuence Analysis Resource (NASQAR; https://nasqar.abudhabi.nyu.edu) was used for creating principal component analysis (PCA) plots of the triplicate samples and a heatmap to visualize the RNA-seq results. A volcano plot was created using ggVolcanoR (https://ggvolcanor.erc.monash.edu). Protein ANNotation with Z-scoRE (PANNZER2; http://ekhidna2.biocenter.helsinki.fi/sanspanz) was used for gene ontology (GO) analysis to predict the genes up- and downregulated in response to erythritol.

Metagenomic DNA was extracted with QIAamp DNA Microbiome Kit (Qiagen, Germany), according to manufacturer’s instructions. The specified kit was chosen because it allows enrichment of the microbial fraction of the sample by differential lysis of eukaryotic cells. Quantity and quality of DNA was assessed using NanoDrop^™ 8,000 Spectrophotometer (Thermo Fisher Scientific, Carlsbad, CA, United States). The A260/280 ratio was in the range of 1.8–1.9, while A260/230 was in the range of 1.8–2.2, reflecting sufficient purity of the DNA preparations. 400 nanograms of DNA from each sample was used as an input for library preparation. DNA was fragmented to 250–320 bp fragments with Covaris ME220 Focused-ultrasonicator (Covaris, Woburn, MA, United States) according to sonication protocol, provided by the manufacturer. The ultrasonic fragmentation method was selected because it minimizes biases in microbial community composition that may arise from suboptimal fragmentase performance for AT or GC-rich genomes. Standard fragment DNBSeq-compatible libraries were prepared using MGIEasy Universal DNA Library Prep kit and further circularized with MGIEasy Circularization Kit (both—MGITech, People’s Republic of China), according to the instructions provided by the manufacturer.

Preparation of NGS libraries and sequencing were conducted by Seibutsu Giken Inc (Japan). The libraries were subjected to multiplexed deep sequencing in 200 bp paired-end mode on the NGS using a BGISEQ-G400 platform (MGI Tech, China). The concentration of the NGS samples was measured using either Synergy LX (Agilent Technologies, Santa Clara, CA) and QuantiFluor dsDNA System (Promega, Madison, WI), or Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and dsDNA HS Assay Kit (Thermo Fisher Scientific). The libraries for DNBSeq were prepared using either MGIEasy FS DNA Library Prep Set (MGI Tech) (25 ng of sample, 8 cycles) or MGIEasy PCR-Free DNA Library Prep Set (MGI Tech) (200 ng of sample, enzymatic digestion for 4 min). The quality of the prepared libraries was assessed using an Agilent 2100 bioanalyzer and a High Sensitivity DNA kit (Agilent Technologies), or Fragment Analyzer and dsDNA 915 Reagent Kit (Agilent Technologies). The DNA nanoball was generated from the libraries using the MGIEasy Circularization Kit (MGI Tech).