human metagenome Metagenome

In 2018, 40 fecal samples from children with diarrhea and 19 fecal samples from healthy controls were collected at The Affiliated Taizhou People's Hospital of Nanjing Medical University in Jiangsu. In addition, 76 fecal samples from children with diarrhea and 27 fecal samples from healthy controls were collected at Children's Hospital of Shanghai. All samples were tested negative for bacteria (including Escherichia, Salmonella, Shigella, Vibrio cholera et al.), mycoplasma, and Chlamydia. All the samples from diarrheal children were tested positive for Rotavirus group A in colloidal gold immunochromatography while all the samples from healthy children negative. Each sample was collected in a 1.5mL sterile centrifuge tube, added 1mL Dulbeccos phosphate-cushioned saline (DPBS). All samples were frozen at -80 Celsius for 30 min and thawed at room temperature for 30 min for three cycles, and then vortexed for 15 min tremendously. The supernatants were collected after centrifugation (5 min, 15,000g, 4 Celsius). Fecal samples were combined into 32 pools (8 diarrheal pools and 4 healthy pools from Taizhou, 15 diarrheal pools and 5 healthy pools from Shanghai) according to the sampling source. Each sample was added 1mL Dulbeccos phosphate-cushioned saline (DPBS) and vortexed for 5min tremendously. The supernatants were collected after centrifugation (5 min, 15,000g). 500 uL of each supernatant was filtered through a 0.45 micron filter (Millipore) to remove eukaryotic and bacterial cell-sized particles. 200 uL of supernatant from each samples was then treated with a mixture of nuclease enzymes to digest unprotected nucleic acids. The remaining total nucleic acids were isolated using a QIAamp Viral RNA Mini Kit (QIAGEN) according to the protocol. Extractions were treated with a reverse transcription kit (SuperScript III Reverse Transcriptase) with six-base random primers to reverse transcribe RNA into cDNA. The Klenow fragment polymerase (New England Biolabs) was then added to synthesize the second strand of cDNA (dsDNA). The resulting dsDNA products were used to construct 32 libraries, using a Nextera XT DNA Sample Preparation Kit (Illumina) and then sequenced using an Illumina NovaSeq platform with 250bp paired ends with dual barcoding for each individual sample pool. Sample collection and all experiments in the present were performed with Ethical Approval given by the Ethics Committee of The Affiliated Taizhou People's Hospital.