title: 16s rRNA Seq Gut Metagenome Young Obese Adults
design: Total DNA was extracted using a modified standard method. Briefly, a stool sample was placed in a 2 ml tube to which 0.1 mm and 0.5 mm diameter glass beads (Sigma, USA) were added in a 3:1 ratio. Then 1 ml of warm lysis buffer (60C) containing 500 mM NaCl, 50 mM Tris-HCl (pH 8.0), 50 mM EDTA and 4% SDS was added. The mixture was vortexed to obtain homogeneity and homogenized for 3 minutes using a MiniLys (Bertin Technologies, USA). The resulting lysate was incubated at 70C for 15 minutes and then centrifuged at 14,000 rpm for 20 minutes. 1 ml of the supernatant was transferred to new tubes and placed on ice. 1 mL of lysis buffer was added to the sediment and the homogenization process was repeated. The supernatants were combined in 15 mL tubes and supplemented with 4 mL of 96% ethanol and 200 mL of 3 M sodium acetate. The mixture was incubated at -20C for at least one hour and then centrifuged at 14,000 rpm at +4C for 15 minutes. The resulting precipitate was washed twice with 80% ethanol, dried at 53C for 30-60 minutes, and dissolved in 200 mL of sterilized MilliQ water. The solution was centrifuged again and transferred to new tubes. RNase A (5 mg/ml) was added to the solution and incubated at 37C for 1 hour. Chloroform was added to the solution at a 1:1 ratio, mixed by vortexing for 1 minute, and centrifuged at 5000g for 5 minutes. The liquid phase was carefully transferred to a new sterile tube and used to perform the PCR. The resulting DNA solution was stored at -20C. Amplification of the V4 variable region of the 16S rRNA gene was carried out in one round using a Verity thermal cycler (Applied Biosystems). PCR products were purified using a Cleanup Mini DNA isolation kit for reaction mixtures (Evrogen, Russia). The concentration of the resulting 16S libraries in solution was determined using a Qubit fluorimeter (Invitrogen, USA) with the Quant-iT dsDNA High-Sensitivity Assay Kit. Purified amplicons were mixed equimolarly based on the obtained concentrations. The quality of the library prepared for sequencing was assessed using agarose gel electrophoresis. Further sample preparation and sequencing of the pooled sample were conducted using the MiSeq Reagent Kit v2 (500 cycles) and the MiSeq instrument (Illumina, USA) according to the manufacturers recommendations. After quality score trimming, DNA read pooling was performed using the SeqPrep package, resulting in a final read length of 252 bp.