Miseq genome sequencer

The feces samples collected from all groups on the third week were used for the microbial community analysis. Total genomic DNAs from the feces of mice were extracted using the E.Z.N.A.® stool DNAKit (Omega Bio-Tek, Norcross, GA, U.S.) according to the manufacturer's instruction. The quality of extracted DNA was checked by 1% agarose gel electrophoresis and spectrophotometry (optical density at the 260 nm/280 nm ratio). All extracted DNA samples were stored at -20°C for further analysis.
The V3–V4 hypervariable regions of the 16s rRNA gene were subjected to high-throughput sequencing by Beijing Allwegene Tech, Ltd. (Beijing, China) using the Illumina Miseq PE300 sequencing platform (Illumina, Inc., CA, USA). The V3-V4 regions of the bacterial 16s rRNA gene were amplified with the universal primers of the forward 338F (5′-ACTCCTACGGGAGGCAGCAG-3) 5and the reverse 806R (5′-GACTACHVGGGTWTCTAAT-3′). The PCR program was as follows: 95°C for 5 min and 25 cycles at 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s with the final extension of 72°C for 10 min. PCR reactions were performed in triplicate: 25 μl mixture containing 2.5 μl of 10× Pyrobest Buffer, 2 μl of 2.5 mM dNTPs, 1 μl of each primer (10 μM), 0.4 U of Pyrobest DNA Polymerase (TaKaRa), and 15 ng of template DNA. The amplicon mixture was applied to the MiSeq Genome Sequencer (Illumina, San Diego, CA, USA).

The nucleotide sequences of the SARS-CoV-2 variants circulated in the Siberian Federal District in the 2020–2022 period were obtained by next-generation sequencing (NGS) in the WHO National Influenza Centre (Research Institute of Influenza) and the Federal Research Center of Fundamental and Translational Medicine [16 (link)], and most of them were deposited in the GISAID database. The selection of samples for sequencing was based on obtaining from several healthcare institutions located in different regions of the Siberian Federal District.
The complete genome NGS sequencing was performed using the Illumina MiSeq platform and the associated reagent kits (Illumina), according to the manufacturer’s methodology (Illumina, Inc. Worldwide Headquarters: 5200 Illumina Way, San Diego, CA 92122, USA). RNA was extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). Whole-genome amplification was performed using the ARTIC primer set [18 ]. DNA libraries were prepared using a Nextera DNA Flex Library Prep kit (Illumina, San Diego, CA, USA). Sequencing of the DNA libraries was conducted on a MiSeq genome sequencer (Illumina) with a reagent kit, version 3 (600-cycle). The consensus sequences were generated using Bowtie 2 software [19 (link)].

Genomic DNA was extracted from 0.5 g soil samples using the E.Z.N.A. ^® Bacterial DNA Kit (Omega Biotek, Norcross, GA, USA) according to the manufacturer’s instructions. DNA quality was tested by 1% agarose gel electrophoresis and spectrophotometry (optical density at 260 nm/280 nm ratio). 16S rRNA gene V3-V4 hypervariable regions were sequenced with Illumina MiSeq PE300 sequencing (Illumina, Inc., San Diego, CA, USA). The pair of universal primers 338F (5′ ACTCCTACGGGAGGCAGCAG-3) and 806R (5′ GACTACHVGGGTWTCTAAT-3′) was used to amplify V3-V4 regions of 16S rRNA. Conditions for the PCR amplification were as follows: 95 °C for 5 min, and 25 cycles at 95 °C for 30 s, 55 °C for 30 s, and at 72 °C for 30 s with a final extension of 72 °C for 10 min. PCR was conducted in triplicate in a 25 μL mixture containing 2.5 μL of 10 × Pyrobest buffer, 2 μL of 2.5 mM dNTPs, 1 μL of each primer (10 μM), 0.4 U of Pyrobest DNA polymerase (TaKaRa), and 15 ng of template DNA. Amplicon mixtures were transfered to the MiSeq Genome Sequencer (Illumina, San Diego, CA, USA).