16S rRNA amplicon sequencing of dog fecal microbiome
DNA was extracted with DNeasy PowerSoil DNA Isolation Kit (Qiagen), the V4 region of the 16S rRNA gene was amplified with a dual-indexing one-step PCR with complete fusion primers (505F/816R) with multiple barcodes, adapted for the Miniseq platform. PCR reactions containing 0.3-30 ng template DNA, 0.1 l Phusion High-Fidelity DNA Polymerase (ThermoFisher), 1X HF PCR Buffer, 0.2 mM each dNTP, and 10 M of the forward and reverse fusion primers were denatured at 98 C for 30 sec, cycled 30 times at 98 C, 10 sec; 55 C, 30 sec; 72 C, 30 sec; incubated at 72 C for 4 min 30 sec for a final extension. PCR products were purified and normalized using the SequalPrep Normalization Kit (ThermoFisher) and pooled into libraries containing 95 samples and 1 negative control. Paired-end sequencing (150 bp) was performed on one mid-output MiniSeq flow cell (Illumina) per final library.