Nucleospin stool kit

DNA was extracted according to the instructions of the NucleoSpin Stool kit, (Macherey Nagel, Germany). DNA concentration and purity were detected using a NanoDrop spectrophotometer, and DNA quality was detected by 1.8% agarose gel electrophoresis. The V3–V4 variable regions of bacterial 16S ribosomal RNA were amplified by PCR with primers 338F and 806R; and the PCR products were mixed in equal proportions, and then purified using a DNA purification column (Omega, American). Sequencing was performed using the Illumina MiSeq platform.

DNA of the choanal and cloacal swabs was extracted using a commercial kit (Genomic DNA from tissue, NucleoSpin^® Tissue from Macherey-Nagel, Düren, Germany) according to manufacturer’s instructions. For each extraction lot, a negative control was prepared by using “Buffer T1” instead of the sample. DNA of the fecal samples was extracted with the NucleoSpin^® Stool kit (Macherey-Nagel, Düren, Germany) according to the company recommendations. Quality (260/280 value) and quantity of extracted DNA was measured using a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNA was stored at −20 °C until further use.