Kapa hifi master mix 2

Rectal swabs were thawed, and DNA was extracted using a QIAmp DNA Stool kit (Qiagen, Hilden, Germany) according to the manufacturer instructions with a minor modification. The rectal swabs were dissolved in 1 ml Buffer ASL and shaken at 1,000 rpm (Mixing Block MB-102, CaRlibiotech S.r.l. Rome, Italy) continuously until the stool samples were homogenized. DNA quality and quantity were assessed with a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA), and the isolated DNA was stored at −20°C until use.
Bacterial DNA was amplified by targeting the V3–V4 hypervariable regions of the 16S rRNA gene (23 (link)). PCR amplification of each sample was performed in a 25-μl volume. A total of 12.5 μl of KAPA HIFI Master Mix 2× (Kapa Biosystems, Inc., MA, USA) were used. Then, 0.2 μl of each primer (100 μM) was added to 2 μl of genomic DNA (5 ng/μl). Blank controls (no DNA template) were also included. Amplification and library quantification were carried out as described previously (24 (link)).

Bacterial DNA was amplified while using the primers that were described in literature [36 (link)], which target the V3-V4 hypervariable regions of the 16S rRNA gene. All of the PCR amplifications were performed in 25 μL volumes per sample. A total of 12.5 μL of KAPA HIFI Master Mix 2× (Kapa Biosystems, Inc., Wilmington, MA, USA) and 0.2 μL of each primer (100 μM) were added to 2 μL of genomic DNA (5 ng/μL). The blank controls (no DNA template added to the reaction) were also performed. A first amplification step was performed in an Applied Biosystem 2700 thermal cycler (ThermoFisher Scientific, Waltham, MA USA). The samples were denatured at 95 °C for 3 min., followed by 25 cycles with a denaturing step at 98 °C for 30 s, annealing at 56 °C for 1 min. and extension at 72 °C for 1 min., with a final extension at 72 °C for 7 min. The amplicons were cleaned with Agencourt AMPure XP (Beckman, Coulter Brea, CA, USA) and libraries were prepared following the 16S Metagenomic Sequencing Library Preparation Protocol (Illumina, San Diego, CA, USA). The libraries obtained were quantified by Real Time PCR with KAPA Library Quantification Kits (Kapa Biosystems, Inc., Wilmington, MA, USA), pooled in equimolar proportion, and then sequenced in one MiSeq (Illumina, San Diego, CA, USA) run with 2 × 250-base paired-end reads.

Bacterial DNA was amplified using the primers that were described in literature [77 (link)], which target the V3-V4 hypervariable regions of the 16S rRNA gene. All the PCR amplifications were performed in 25 µL volumes per sample. A total of 12.5 µL of KAPA HIFI Master Mix 2× (Kapa Biosystems, Inc., Wilmington, MA, USA) and 0.2 µL of each primer (100 µM) were added to 2 µL of genomic DNA (5 ng/µL). The blank controls (no DNA template added to the reaction) were also performed. A first amplification step was performed in an Applied Biosystem 2700 thermal cycler (ThermoFisher Scientific, Waltham, MA USA). The samples were denatured at 95 °C for 3 min, followed by 25 cycles with a denaturing step at 98 °C for 30 s, annealing at 56 °C for 1 min, and extension at 72 °C for 1 min, with a final extension at 72 °C for 7 min. The amplicons were then cleaned with Agencourt AMPure XP (Beckman, Coulter Brea, CA, USA), and libraries were prepared following the 16S Metagenomic Sequencing Library Preparation Protocol (Illumina, San Diego, CA, USA). The libraries obtained were quantified using real-time PCR with KAPA Library Quantification Kits (Kapa Biosystems, Inc., Wilmington, MA, USA), pooled in equimolar proportion and then sequenced in one MiSeq (Illumina, San Diego, CA, USA) run with 2 × 250-base paired-end reads.