Qiasymphony powerfecal pro dna kit

Cells were recovered from filters followed by DNA isolation using the Qiagen QIAsymphony PowerFecal Pro DNA Kit (Qiagen, Hilden, Germany) [46 ], which involved cellular lysis through mechanical disruption and enzymatic treatment. Subsequently, DNA purification from the sample was performed using a silica/gel column, allowing for DNA isolation and the removal of contaminants and inhibitors for future reactions using the QIAamp DNA Micro Kit (Qiagen, Hilden, Germany) [47 ]. Cellular lysis was carried out through mechanical disruption and enzymatic treatment. Subsequently, DNA purification from the sample was performed using a silica/gel column, enabling DNA isolation and the removal of contaminants and inhibitors for future reactions using the Qiagen QIAamp DNA Micro Kit. The quality and concentration of the DNA were evaluated using Nanodrop.

Metataxonomic analysis was performed on fecal samples collected from all 235 patients of the PROBE-IBS/2 cohort and 100 control samples from healthy subjects by profiling the 16S rRNA gene. Fecal samples were collected by the patients and delivered to the reference hospital within 24 h with instructions to store them in a refrigerator (without freezing) before delivery. Subsequently, the samples were promptly frozen at −80°C and ultimately transferred in dry ice to the centralized laboratory at the University of Milan for analysis. Total DNA was extracted from 150 mg of feces using the QIAsymphony PowerFecal Pro DNA Kit (Qiagen, Milan, Italy) in accordance with the manufacturer’s instructions. To amplify a fragment of the 16S rRNA gene that includes the V3 and V4 variable regions, primers 341F (5’-CCT ACG GGN GGC WGC AG-3’) and 805 R (5’-GAC TAC HVG GGT ATC TAA TCC-3’) (LC Sciences, Houston, TX) were used. The obtained amplicons were sequenced using the NovaSeq 6000, 2 × 250bp (NovaSeq 6000 SP Reagent Kit, 500 cycles). The resulting sequencing reads were analyzed using the bioinformatic pipeline Quantitative Insights Into Microbial Ecology (QIIME) 2 version 2022.2, with the Greengenes database v. 13_8 for taxonomic assignment to amplicon sequence variants (ASVs) clustered at a 97% similarity (cASV) using the Divisive Amplicon Denoising Algorithm (DADA2; 27214047).