Maxwell 16 dna purification kit

Fecal samples were collected before and 6 months after BIB. Fifty milli gram of feces were homogenized in 500 μl of 10 mg/ml lysozyme solution in Tris-sucrose buffer (50 mM Tris-HCl, 40 mM EDTA, 0.75 M sucrose, pH 8.0) and incubated for 1 h at 37°C. The DNA was purified using the Maxwell® 16 DNA Purification Kit and the Maxwell® 16 Instrument (Promega, Madison, WI, USA), according to the manufacturer's instructions. The DNA quality was checked by agarose gel electrophoresis and the DNA concentration was determined using the Qubit HS dsDNA fluorescence assay (Life Technologies, Carlsbad, CA, USA).

Fecal samples were refrigerated within 24 h after collection, and aliquots were stored at -80°C until analysis. According to the manufacturer’s instructions, DNA was extracted using the Maxwell^® 16 DNA purification kit and the protocol was carried out on the Maxwell^® 16 Instrument (Promega, Madison, WI, USA). We used the primers and workflow to generate the amplicon from the V4 region of the 16s rRNA gene according to Penington et al. (2018 (link)). The amplicon library produced was sequenced on the Illumina MiSeq platform (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions.
The raw read files were processed in the R environment using the dada2 package [10.1038/nmeth.3869] (Ombrello, 2020 (link)). The forward and reverse sequences were trimmed to 150 bases. Reads containing more than two expected errors were removed. Errors in filtered sequences were corrected by the algorithm and joined to form the amplicon sequence variants (ASVs). The chimeric sequences were also removed, and a sample count table was generated. The taxonomic classification was done with the tag.me package [10.1101/263293] using the model 515F-806R (Pires et al., 2018 (link)).