DB32T 3762.11-2020新型冠状病毒检测技术规范 第11部分：全基因组高通量测序

ICS11.020C62江苏省地DB32方标准DB/T3762.11—2020TechnicalspecificationsforSARS-CoV-2detectionPartWholegenomehigh-throughputsequencing2020-07-29发布2020-07-30实施江苏省市场监督管理局发布前言DBT。GB/T1.1-2009给出的规则起草。江苏省卫生健康委员会提出。。于新型冠状病毒全基因组高通量测序。范性引用文件凡是不注日期的引用文件，其最新版本(包括所有的修改单)适用于本文件。GB489实验室生物安全通用要求状病毒实验室生物安全指南中华人民共和国国家卫生健康委员会原卫生部办公厅实验室工作导则原卫生部检验中心原国家卫生计生委定义文件wholegenomesequencing生物体整个基因组序列进行测定，以获得完整的基因组信息。高通量测序high-throughputsequencing("next-generation"sequencing)或深度测序(deepsequencing)技术。eadsads度sequencingdepth的总碱基数与待测基因组大小的比值。sequencingcoverage的序列占整个基因组的比例。4环境与设施4.1实验室资质及级别要求二级实验室(BSL-2)进行，同时采用生物安全三级实验室(BSL-3)的个人防护；感染性材料或活病毒在采用可靠方法灭活后进行的核酸检验实验室工作导则》相关要求。4.2操作人员资质及防护要求设备与耗材5.1设备R5.2耗材试剂cDNA成试剂盒、随机六聚体引物、经验证可靠的适理与核酸提取DBT3762.3的第7章进行。板制备8.1cDNA制备一链cDNA。混合引物污染和降解。RPCR产物的纯化PCRPCRmMEDTA)洗脱纯化后的产物。DNA文库构建9.1纯化产物的定量与标化9.2DNA文库制备与混合使用的建库试剂盒说明书进行文库制备、定量和文库混合。根据高通量测序试剂盒和高通量测序仪操作说明完成试剂装载，设置测序参数进行测序。大引物长度30bp进行Trim)和低质量的Reads，然后以SARS-CoV-2参考基因组(GeneBank号：MN列mapping，mapping之后对测序覆盖度和测序深度进行分析，最终生成一致性(consensus)序列，即为待测样本的全基因组序列。A(规范性附录)引物序列(5′-3′)1Pool1AACCAACTTTCGATCTCTTGT2Pool1GCCATCTTTAAGATGTTGACG3nCoV-2019_2_LEFTPool2CAGGTTCGCGACGT4nCoV2019_2_RIGHTPool2CAGTGCCAAGCTCGT5nCoV-2019_3_LEFTPool1AAGGAGCTGGTGGC6nCoV2019_3_RIGHTPool1GGTGTCTGCAATTCATAGCTCT7nCoV-2019_4_LEFTPool2ACTGCTGCCGTGAAC8nCoV2019_4_RIGHTPool2AGTGGCACCTTCTTTAGT9nCoV-2019_5_LEFTPool1ACTTCATGGCAGACGnCoV2019_5_RIGHTPool1TGTTGACTTTCTCTTTTTGGAGTnCoV-2019_6_LEFTPool2GTTGGAGAAGGTTCCGnCoV2019_6_RIGHTPool2CCTTCTGTAAAACACGnCoV-2019_7_LEFTPool1CAGAGGCTGCTCGTGTTGTAnCoV2019_7_RIGHTPool1GGTGACAATTTGTCCAnCoV-2019_8_LEFTPool2AGTTTCTTAGAGACGGTTGGGAnCoV2019_8_RIGHTPool2GCTTCACTAGTAGGTnCoV-2019_9_LEFTPool1GAAGTGTTAACAGAGGAnCoV2019_9_RIGHTPool1CAGCATCTGCCACAAPool2GTGCTCTGCCTATACAGTPool2AACCAATCTTCTTCTTGCTCTPool1GTGCCACTTCTGCTPool1TTCAACTTGCATGGCAPool2ACATGGAGGAGGTGTTGCAGPool2CTTCATTTCCAAAAAGCTTGAPool1CAAATGTCTACTTAGCTGTPool1AACCACAGCAGTTAAAACACPool2ATTCTGCCACTCTTGTPool2TTCCACACAGACAGGCATTPool1GTGCTTAAAAAGTGTAAAAGTGCCPool1AAACAGAAACTGTAGCTGGCPool2TTGGAAGAAGCTGCTCGGTPool2TGCGTGTGGAGGTTAPool1CTTTGAGAGAAGTGAGGACTPool1GAGTGTTAACAATGCAGTPool2ACCCACAAGTTAATGGTTTAAC引物序列(5′-3′)Pool2TGTTTACCACACGTACAAGGPool1TGTACATGGGCACACTPool1CTTAGGGTCAATTTCTGTnCoV2019_20_LEFTPool2AAAGAAAACAGTTACACAACAACCA0nCoV019_20_RIGHTPool2GTGGCTTTATTAGTTGCATTGTT1nCoV2019_21_LEFTPool1TTGATTATAAACACTACACA2nCoV019_21_RIGHTPool1TGTGTGGCCAACCTCTnCoV2019_22_LEFTPool2CCGAAGTTGTAGGAGACATTATACT4nCoV019_22_RIGHTPool2AGTATTCTTTGCTATAGTAGTCGGC5nCoV2019_23_LEFTPool1ACTACTAACATAGTTACACGGTGT6nCoV019_23_RIGHTPool1AGTACAGTAGGTTGCAATAGTG7nCoV-2019_24_LEFTPool2CATGCCTTCTTACTGTACTG8nCoV019_24_RIGHTPool2TTCTAACCATAGCTGAAATCGGG9nCoV2019_25_LEFTPool1TTTTCAGCTATTTTGCAGTnCoV019_25_RIGHTPool1TGTAGTGACAAGTCTCTCGCAnCoV2019_26_LEFTPool2TACATTCTGTGCTGGTAGTnCoV019_26_RIGHTPool2CTATCACCAACATCAGnCoV2019_27_LEFTPool1AGTCAGCTTATGTGTCAACCnCoV019_27_RIGHTPool1TACAAGCACCAAGGTCACGGnCoV2019_28_LEFTPool2TAGAAGTTACTGGCGATAGTTGTnCoV019_28_RIGHTPool2GACATGACATGAACAGGTGTnCoV2019_29_LEFTPool1TTGTGTTCCTTTTTGTTGCTGCnCoV019_29_RIGHTPool1TGTACTCTATAAGTTTTGATGGTGTGTnCoV2019_30_LEFTPool2CTAATGGTGACTTTTTGCAnCoV019_30_RIGHTPool2CTAGTAGATACACAAACACCAGnCoV2019_31_LEFTPool1AGTACTGTAGGCACGGCnCoV019_31_RIGHTPool1AATAAACACCAGGTAAGAATGAGTnCoV2019_32_LEFTPool2TACAGTCATGTAGTTGCCnCoV019_32_RIGHTPool2GCACATCACTACGCAACnCoV2019_33_LEFTPool1TTTTGAAGAAGCTGCGCTGTnCoV019_33_RIGHTPool1AAACTACGTCATCAAGCnCoV2019_34_LEFTPool2CTGGTAAAGTTGAGGGTnCoV019_34_RIGHTPool2GAAATTGGGCCTCATAGCAnCoV-2019_35_LEFTPool1CATTCAACCAGGACAGnCoV019_35_RIGHTPool1TCATAGCCACAAGGTTAAAGTCAnCoV2019_36_LEFTPool2TACAGTTAATGTTTTAGCTTGGTTGTAnCoV019_36_RIGHTPool2AGACCATTGAGTACTCTGGAnCoV2019_37_LEFTPool1CACCACTGGTTGTTACTCACnCoV019_37_RIGHTPool1ACTCTCCTAGCACCATnCoV2019_38_LEFTPool2GTGTTATGTATGCATCAGCTGTnCoV019_38_RIGHTPool2TAAACACCAAGAGTCAGTCTAAA引物序列(5′-3′)nCoV2019_39_LEFTPool1TTGCCCTATTTTCTTCATAACTGGTnCoV019_39_RIGHTPool1TGGACACATTGAGCCCnCoV2019_40_LEFTPool2CAGTAGTCTTACTCTCAGTnCoV019_40_RIGHTPool2CATCACGGTCAAATnCoV2019_41_LEFTPool1CCATCATATGCAGCTnCoV019_41_RIGHTPool1CAACCATTAGTTTGGCTnCoV2019_42_LEFTPool2GAGATGGTTGTGTTCCCnCoV019_42_RIGHTPool2CCTTTGTTGTGTTGTnCoV2019_43_LEFTPool1GATGTCTTGTGCTGCnCoV019_43_RIGHTPool1AGCATCTACAGCAAAAGCAnCoV2019_44_LEFTPool2GTACGTCTACAAGCTnCoV019_44_RIGHTPool2CCTTTCCACATACCGCAGACnCoV2019_45_LEFTPool1CTTGTGCTAATGACCCnCoV019_45_RIGHTPool1ATTGTTTCTTCATGTTGGTAGTTAGAGAnCoV2019_46_LEFTPool2CCAAGAAAAGGACGnCoV019_46_RIGHTPool2ACCTAAGTTGGCGTAnCoV2019_47_LEFTPool1TGGTATGATTTTGTAGAAAACCCnCoV019_47_RIGHTPool1AACGGTCAAAGAGTTTTAACCTCTCnCoV2019_48_LEFTPool2ACACTGACTTAACAAAGCCTnCoV019_48_RIGHTPool2TACCAGAAGCAGCGTGCnCoV2019_49_LEFTPool1AATTACTTGTGTATGCTGCTGAnCoV019_49_RIGHTPool1TGACTTGGTTAGCATTAATACAnCoV2019_50_LEFTPool2GTACTTTGATTGTTACGATGGTnCoV019_50_RIGHTPool2GTTGTGCCAACCACCAnCoV2019_51_LEFTPool1CCGCCACTAGAGGAGnCoV019_51_RIGHTPool1TGCATTAACATTGGCCGTGAnCoV2019_52_LEFTPool2AGATGCCACAACTGCnCoV019_52_RIGHTPool2AAATTCATGAGGTCCnCoV2019_53_LEFTPool1AAATGTTGGACTGAGACTGAnCoV019_53_RIGHTPool1CTCATAAAACTCAGGTTCCCnCoV2019_54_LEFTPool2CAGGACACATGTTAGACAnCoV019_54_RIGHTPool2CAAAAACTTGTCCATTAGCACAnCoV2019_55_LEFTPool1CAACTTTACTTAGGAGGTATGAGCTnCoV019_55_RIGHTPool1CTCCTATTTGTACTTTACTGTnCoV2019_56_LEFTPool2TAGACCACCACTTAACCGAnCoV019_56_RIGHTPool2ACTATGCGAGCAGAAGGGTAnCoV-2019_57_LEFTPool1TCTACACTCCAGGGACCACCnCoV019_57_RIGHTPool1GCAGGGTCGCCAATnCoV2019_58_LEFTPool2GAGTGTTGTCAATGCCAGAnCoV019_58_RIGHTPool2CAAGCAGGGTTACGTnCoV2019_59_LEFTPool1ATGATGTTTCATCTGCA引物序列(5′-3′)nCoV019_59_RIGHTPool1AGTCCTGTTACATTTTCAGCTTGnCoV2019_60_LEFTPool2AGACCTTTATGACAAGTTGCAnCoV019_60_RIGHTPool2ACAGCTTCTCTAGTAGCnCoV2019_61_LEFTPool1TCACCCGCGAAGAAGCnCoV019_61_RIGHTPool1CACATAGACAACAGGTGCGCnCoV2019_62_LEFTPool2GGCTTTGAGTTGACAnCoV019_62_RIGHTPool2TTTCTACAAGCCGCnCoV2019_63_LEFTPool1AGCGTGTTGACTGGACTnCoV019_63_RIGHTPool1ACTGCCACCATCACAACCnCoV2019_64_LEFTPool2ATATCCTGCTAATTCCATTGTnCoV019_64_RIGHTPool2TTGTAAAAGTGTTCCAGAGGTnCoV2019_65_LEFTPool1TAGCTTGTGGGTTTnCoV019_65_RIGHTPool1CATAGAACAAACACCAATAGTnCoV2019_66_LEFTPool2GACATTGCTGCTnCoV019_66_RIGHTPool2CATTTGACTCCTGGGTnCoV2019_67_LEFTPool1AACAATTACCTGAAACTTACTnCoV019_67_RIGHTPool1GAAACTACAGATAAATCTTGGGnCoV2019_68_LEFTPool2GTTCATCTAAGTGTGTGTGTnCoV019_68_RIGHTPool2ATCAGAACCAGCACCAnCoV2019_69_LEFTPool1AAAATATACTCAACTGTGTCAnCoV019_69_RIGHTPool1AGCCACGGAACCTCCAnCoV2019_70_LEFTPool2AAGAAAATGACTCTAAAGAGGGTTTnCoV019_70_RIGHTPool2TCTTTTAAAGACATAACAGCAGnCoV2019_71_LEFTPool1AATCCAATTCAGTTGTCTTCCTATTCnCoV019_71_RIGHTPool1AAAGGTAAGAACAAGTCCTnCoV2019_72_LEFTPool2CGTGGTGTTTATTACCCTGACnCoV019_72_RIGHTPool2CTGAACTCACTTTCCATCCAACnCoV2019_73_LEFTPool1AATGATCCATTTTTGGGTGTnCoV019_73_RIGHTPool1TGTCCAACCTGAAGAnCoV2019_74_LEFTPool2CATCACTAGGTTTCAAACTTTACTTGCnCoV019_74_RIGHTPool2CAGTTGCTGATTCTCTTCnCoV2019_75_LEFTPool1GAGTCCAACCAACAGAATCTATTGTnCoV019_75_RIGHTPool1ACCAACCTTAGAATCAAGATTGTnCoV2019_76_LEFTPool2GGCAAACTGGAAAGATTGCTnCoV019_76_RIGHTPool2CCAAATTGTTGGAAAGGCAnCoV2019_77_LEFTPool1CTGTTTGTGGACCTAnCoV019_77_RIGHTPool1TATTAAACAGCCTGCnCoV2019_78_LEFTPool2TCCTACTTGGCGTGTnCoV019_78_RIGHTPool2CAAAAACTGCCATATTGCAnCoV2019_79_LEFTPool1TTCAACTGAATGCAGCnCoV019_79_RIGHTPool1TCTGTGAGCAAAGGTGG_________________________________引物序列(5′-3′)nCoV2019_80_LEFTPool2TGGTGATATTGCTGCTnCoV019_80_RIGHTPool2TAAGTTGTTTAACAAGCGnCoV2019_81_LEFTPool1AAAACTTCAAGATGTGGnCoV019_81_RIGHTPool1TTTTCTTGTGCAGGGnCoV2019_82_LEFTPool2CATCTTATGTCCTTCCCTnCoV019_82_RIGHTPool2GATGTCACCTAAATCAAnCoV-2019_83_LEFTPool1CAACCTGAATTAGACTCAnCoV019_83_RIGHTPool1TCCTTTGAGCACTGGCnCoV2019_84_LEFTPool2GTTGTCTCAAGGGCTnCoV019_84_RIGHTPool2GGTGTGAGTAAACTGTTACAAACAACnCoV2019_85_LEFTPool1TAGCACTCTCCAAGGGTGTTnCoV019_85_RIGHTPool1GTCTTTTACTCCAGATTCCCnCoV2019_86_LEFTPool2GATGGCACAACAAGTCnCoV019_86_RIGHTPool2AAAGCAAGAAAAAGAAGTACGCnCoV2019_87_LEFTPool1AGCGTGCCTTTGTAnCoV019_87_RIGHTPool1GGTTCCATTGTTCAAGGAG