16S ribosomal gene amplicon of gut microbiota of Trogopterus xanthipes
Bacterial genomic DNA was extracted from fecal samples (200 mg) using QIAamp  Fast DNA Stool Mini kit (QIAGEN, Valencia, CA, USA) following pathogen detection protocols. The V3-V4 region of the bacterial 16S rRNA gene was PCR-ampli ed using barcoded forward primers (XXXXXXCCTACGGGNGGCWGCAG) and reverse primers (XXXXXXGACTACHVGGGTATCTAATCC); 6-bp barcodes were indicated by XXXXXX. The PCR was performed under the following conditions: 94 C for 4 min, followed by 25 cycles of 94 C for 30 s, 57 C for 30 s, and 72 C for 30 s, with a  nal elongation step at 72 C for 8 min. Amplified products of expected size (464 bp) were purified with QIAquick Gel Extraction Kit (QIAGEN) and DNA concentration determined using Qubit  3.0 Fluorometer (Invitrogen, San Diego, CA, USA). Three amplicon libraries were constructed by pooling 20 samples with equal amounts of DNA for each library. The 16S rRNA gene amplicons were pair-ended sequenced (2 x 301 bp) with 1 Gb qualified outputs per library by Illumina MiSeq platform in Yourgene Bioscience Company (Taiwan).