In this study, fecal samples were collected from both wild and captive giant pandas. Fecal samples were collected from wild giant pandas at the Fengtongzhai National Nature Reserve (FNNR, n = 14) and the Wolong National Nature Reserve (WNNR, n = 67) by experienced trackers, immediately frozen in liquid nitrogen, and stored at −80 °C until later use. Fecal samples from captive giant pandas were collected from the China Conservation and Research Center for the Giant Panda (CCRC; n = 49) and also stored at −80 °C until later use. Demographics for all pandas sampled in this study are shown in Table S1 . Seven tetra-microsatellites including GPL-60, gpz-47, gpz-20, GPL-44, GPL-29, GPL-53, and gpz-6 were used to distinguish the wild individuals, and this DNA analysis was performed by Qiao et al., therefore the samples of wild giant pandas were from their research (Table S2 ) [25 (link)].
DNA was extracted from the fecal samples using the Mo Bio PowerFecal DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. Variable region 4 (V4) forward primer: GTGYCAGCMGCCGCGGTAA, reverse primer: GGACTACHVGGGTWTCTAAT. The PCR reaction (50 µl total volume) contained 2 µL DNA (20 ng), 19 µL PCR grade water, 25 µL 2× Es Taq Master Mix (CW BIO, Beijing, China) (including 2× Es Taq Polymerase, 0.075 µM Mg2+, and 10 µM dNTP mix), 2 µL (0.4 µM) forward primer, and 2 µL (0.4 µM) reverse primer. PCR was performed at 94 °C for 1 min, followed by 30 cycles of 94 °C for 20 s, 59 °C for 25 s, and 68 °C for 45 s, followed by a final extension at 68 °C for 10 min. Variable region 4 was sequenced at the Beijing Genomics Institute (Beijing, China).
Shotgun metagenomic sequencing was performed at Novogene (Beijing, China). DNA libraries were constructed according to Illumina’s instructions. Briefly, DNA was sheared to 300–400 bp fragments, and the DNA from each individual sample was barcoded uniquely. Since most of the demographic data for the wild pandas are unknown, we intended to choose a wider range of samples from the captive samples to see if the environment is still the major driver of the gut metagenome. Therefore, we randomly chose three cubs (≤2-year-olds), one sub-adult (>2-year-olds and <5-year-olds) and three adults (≥5-year-olds and ≤20-year-olds) (including three males and four females) for metagenome analysis (Table S1 ). Correspondingly, seven random samples were collected from wild giant pandas at the Fengtongzhai National Nature Reserve and were chosen for comparative analysis of metagenomics with captive giant pandas. Fourteen samples were sequenced on an Illumina HiSeq platform. Sequencing depth, quality control, and pre-processing details are listed in Table S3 .
The datasets used in this study are accessible from the National Centre for Biotechnology Information Sequence Read Archive (SRA;http://www.ncbi.nlm.nih.gov/sra ) [26 (link)], accession bio project numbers: PRJNA356809 and PRJNA358755.
DNA was extracted from the fecal samples using the Mo Bio PowerFecal DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA, USA) according to the manufacturer’s instructions. Variable region 4 (V4) forward primer: GTGYCAGCMGCCGCGGTAA, reverse primer: GGACTACHVGGGTWTCTAAT. The PCR reaction (50 µl total volume) contained 2 µL DNA (20 ng), 19 µL PCR grade water, 25 µL 2× Es Taq Master Mix (CW BIO, Beijing, China) (including 2× Es Taq Polymerase, 0.075 µM Mg2+, and 10 µM dNTP mix), 2 µL (0.4 µM) forward primer, and 2 µL (0.4 µM) reverse primer. PCR was performed at 94 °C for 1 min, followed by 30 cycles of 94 °C for 20 s, 59 °C for 25 s, and 68 °C for 45 s, followed by a final extension at 68 °C for 10 min. Variable region 4 was sequenced at the Beijing Genomics Institute (Beijing, China).
Shotgun metagenomic sequencing was performed at Novogene (Beijing, China). DNA libraries were constructed according to Illumina’s instructions. Briefly, DNA was sheared to 300–400 bp fragments, and the DNA from each individual sample was barcoded uniquely. Since most of the demographic data for the wild pandas are unknown, we intended to choose a wider range of samples from the captive samples to see if the environment is still the major driver of the gut metagenome. Therefore, we randomly chose three cubs (≤2-year-olds), one sub-adult (>2-year-olds and <5-year-olds) and three adults (≥5-year-olds and ≤20-year-olds) (including three males and four females) for metagenome analysis (
The datasets used in this study are accessible from the National Centre for Biotechnology Information Sequence Read Archive (SRA;
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