Nextera xt fragmentation enzyme

Each fecal sample was collected in a clean, dry screw-top container and stored directly at −80 °C. All the samples were then transferred to the study laboratory and kept at −80 °C for further analysis. Later, the DNA was extracted from 0.25 g frozen stool aliquots using the QIAGEN PowerFecal DNA Kit (Catalogue: 12830-50). The purity of the isolated DNA (260/280 ratio) and its concentration was measured using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The DNA concentration was ≥1.60. DNA libraries were prepared using the Nextera XT DNA Library Preparation Kit (Illumina) and Nextera Index Kit (Illumina), with a total DNA input of 1 ng. Genomic DNA was fragmented using a proportional amount of Illumina Nextera XT fragmentation enzyme. Combinatory dual indices were added to each sample, followed by 12 cycles of PCR to construct the libraries. The DNA libraries were purified using AMpure magnetic beads (Beckman Coulter) and eluted in QIAGEN EB buffer. The samples were sequenced on an Illumina HiSeq 4000, 2 × 150 bp. DNA extractions were sent to CosmosID (Rockville, MD, USA) to identify the gut microbiota composition at the level of the main microbial phyla by identifying the total bacterial DNA and the Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, Verrucomicrobia, and Fusobacteria DNA using the WGS metagenomic sequencing technique.

Following the manufacturer’s protocol, using the QIAGEN DNeasy PowerSoil Pro Kit (Qiagen, Germantown, MD, United States), DNA from samples was isolated. Quantification of the extracted DNA samples was done using Qubit 4 fluorometer and Qubit™ dsDNA HS Assay Kit (Thermofisher Scientific, MA, United States).
Preparation of DNA libraries was done using the Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, United States) and IDT Unique Dual Indexes with total DNA input of 1 ng. Using a proportional amount of Illumina Nextera XT fragmentation enzyme, genomic DNA was fragmented. To each sample, unique dual indexes were added and then libraries were constructed after 12 cycles of PCR. With AMpure magnetic Beads (Beckman Coulter, Brea, CA, United States), DNA libraries were purified and eluted in QIAGEN EB buffer. Libraries were then sequenced on an Illumina NovaSeq 6000 System with S4 Flow Cell.