16s of cat urine: adult spayed female
the hypervariable regions 3 and 4 (V3-V4) of the bacterial 16S rRNA gene was amplified from each urine and NCS extracts and sequenced using a custom protocol developed for the Illumina MiSeq sequencer platform (San Diego, CA, USA) by the BioFab Research (Rome, Italy). The bacterial 16S rRNA gene amplicon library was generated via PCR amplification of V3-V4 hypervariable regions carried out with the KAPA HiFi HotStart DNA Polymerase (Roche Sequencing Solutions, Pleasanton, CA, USA) and the universal primers Bakt_341F (CCTACGGGNGGCWGCAG) and Bakt_805R (GACTACHVGGGTATCTAATCC) complemented with the Illumina adapter sequences. Each sample was amplified with 25 PCR cycles through the following conditions: 95 C for 30 seconds, 55 C for 30 seconds and 72 C for 30 seconds. Negative control in the form of a PCR-amplified ultrapure water sample was included in the reaction. Amplicons were analysed by gel electrophoresis, purified and the DNA concentration of the eluted product was determined by Qubit fluorometer machine (Thermo Fisher Scientific, Life Technologies). DNA samples were sequenced by Illumina MiSeq sequencer platform using a paired-end 2 x 300-bp reagent cartridge according to the manufacturers instructions (MiSeq Reagent Kit v3, Illumina, San Diego, CA, USA). PCR amplification and sequencing of all urine samples and NCS were performed simultaneously in the same run to avoid introducing variability between different reactions.