human metagenome Genome sequencing. human 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.

2018年，研究团队于江苏省南京医科大学附属泰州人民医院收集了40份腹泻儿童粪便样本与19份健康对照儿童粪便样本；同时在上海儿童医院收集了76份腹泻儿童粪便样本与27份健康对照儿童粪便样本。所有样本经检测均排除细菌（包括埃希菌属、沙门菌属、志贺菌属、霍乱弧菌等）、支原体及衣原体感染。腹泻儿童样本经胶体金免疫层析法（colloidal gold immunochromatography）检测均呈A组轮状病毒阳性，健康儿童样本检测结果均为阴性。

每份样本置于1.5mL无菌离心管中，加入1mL杜氏磷酸盐缓冲液（Dulbecco's phosphate-buffered saline, DPBS）。将所有样本经-80℃冷冻30分钟、室温融化30分钟循环三次，随后剧烈涡旋振荡15分钟。经离心（5分钟，15000g，4℃）后收集上清液。按照采样来源将上述粪便样本合并为32个样本混合池（泰州地区8个腹泻样本混合池、4个健康对照样本混合池；上海地区15个腹泻样本混合池、5个健康对照样本混合池）。

针对每个样本混合池，加入1mL杜氏磷酸盐缓冲液（DPBS）并剧烈涡旋振荡5分钟，经离心（5分钟，15000g）后收集上清液。取每份上清液500μL经0.45μm滤器（密理博Millipore）过滤，以去除真核及细菌尺寸的颗粒。随后取每份样本上清液200μL，用核酸酶混合物处理以消化未受保护的核酸。采用QIAamp病毒RNA迷你试剂盒（QIAamp Viral RNA Mini Kit, QIAGEN）按实验流程分离剩余的总核酸。提取产物经搭载六碱基随机引物的SuperScript III反转录试剂盒（SuperScript III Reverse Transcriptase）处理，将RNA反转录为互补DNA（cDNA）。随后加入克列诺片段聚合酶（Klenow fragment polymerase, New England Biolabs）合成cDNA第二链，得到双链DNA（dsDNA）产物。使用Nextera XT DNA样本制备试剂盒（Nextera XT DNA Sample Preparation Kit, Illumina）构建32个测序文库，随后采用Illumina NovaSeq测序平台，针对每个独立样本混合池开展带有双条形码标记的250bp双端测序。

本研究的样本收集及所有实验操作均获得南京医科大学附属泰州人民医院伦理委员会的伦理批准。