Ion pgmtm hi q sequencing kit

Microbial DNAs from the fecal samples were extracted from approximately 200 mg of each stool sample using the QIAamp PowerFecal Pro DNA kit (Qiagen, Valencia, CA, USA) by following the manufacturer’s protocol. To construct sequencing libraries, the V4 to the V5 hypervariable region in the 16S rRNA gene was amplified using the 515 F (5′-barcode-GTGCCAGCMGCCGCGGTAA-3′) and 907 R (5′-barcode-CCGYCAATTCMTTTRAGTTT-3′) indexed reverse primers. The specific PCR conditions are described in a previous study [32 (link)]. Afterward, gel electrophoresis and a Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) were used to confirm the quality and quantity of the library DNA. The library DNAs were then pooled equally and then purified using an AMPure XP bead (Beckman Coulter, Brea, CA, USA). The libraries were sequenced using an Ion Torrent PGM platform for 1250 flows with the Ion PGM^TM Hi Q Sequencing Kit (Thermo Fisher Scientific, USA).

DNA extraction was performed using the DNeasy Plant Mini Kit (QIAGEN, Venlo, the Netherlands). We used the universal primer pair g and h designed by Taberlet et al.^{38 (link)} to amplify the DNA region of the chloroplast trnL P6 loop. Due to its high conservativeness, moderate mutation rate of amplicon sequences and short fragment sizes (usually below 150 bp), the g-h primer pair is widely used in metabarcoding diet analyses of herbivores^{2 (link),39 (link)} as universal primer that targets vascular plants. To track the resulting sequences from each sample, the forward primer was tagged with a multiplex identifier (MID)^{40 (link)}. The detailed procedure of amplicon library preparation is described in Ando et al.^{5 (link),10 (link)}. We performed one run of amplicon sequencing using an Ion Torrent Personal Genome Machine (PGM) system with the Ion PGM^TM Hi-Q Sequencing Kit and the Ion 318^TM Chip (Thermo Fisher Scientific) following the manufacturer’s instructions.