血浆宏病毒组学中降低非病毒核酸方法的探索研究  

Different strategies of reducing non-viral nucleic acid of meta-virome in plasma:a comparative study

作　　者：刘安庆 杨春晖[1] 赵玉伟[2] 赵梦伊 高瞻[1] 黄杨 何苗[1] LIU Anqing;YANG Chunhui;ZHAO Yuwei;ZHAO Mengyi;GAO Zhan;HUANG Yang;HE Miao(Institute of Blood Transfusion,Chinese Academy of Medical Sciences&Peking Union Medical College,Chengdu 610052,China;Chengdu Blood Center)

机构地区：[1]中国医学科学院北京协和医学院输血研究所,610052 [2]成都市血液中心

出　　处：《中国输血杂志》2023年第5期388-395,共8页Chinese Journal of Blood Transfusion

基　　金：四川省科技厅重点研发项目(2019YFS0319);非编码RNA与药物四川省高校重点实验室研究基金(FB20-03);四川省国际科技创新合作/港澳台科技创新合作项目(2021YFH0063)。

摘　　要：目的探究常用的降低非病毒核酸方法对血浆病毒组丰度的影响。方法应用3种建库流程、5种离心条件、2种滤器、4种酶和4种含量氯仿处理定量投入伪狂犬病病毒(PRV)和猪细小病毒(PPV)各2.16 mL的模拟宏病毒组血浆标本共21.6 mL,累计54份标本,随后提取病毒核酸、用qPCR定量检测线粒体DNA(mtDNA)和病毒、构建二代测序(NGS)文库并应用NGS测序仪测序,测序数据用Kraken Py2.0软件做比对并做物种注释分析,得到每个片段对应的物种分类信息以分析降低非病毒核酸的不同方法对微生物和2种指示病毒相对丰度的影响。结果NGS测序仪测序:306.27 GB raw data,193.17 GB clean data,所有的测序数据Q20>90%、Q30>85%,碱基错误率0.03%,GC含量中位数为45.02%。DNA建库流程提高了微生物序列占比和PRV丰度[(91.8±0.5)%](P<0.05);RNA建库和合并建库提高了RNA病毒——瘟病毒(Pestivirus)的丰度,PRV丰度分别为(17.7±3.3)%和(8.1±1.5)%。5种离心条件处理后mtDNA Ct值增加、人类序列降低至占比<(89.5±1)%、微生物序列升高至占比>(2.4±0.03)%(P<0.05);在4℃、100 g离心30 min后PRV丰度提升至(40.6±6)%,再于4℃、4000 g离心45 min后PRV丰度下降至(4.1±0.01)(P<0.05)。依次使用0.22和0.45μm滤器使mtDNA增加至Ct值>25.56±0.13,人类序列降低至占比<(86.1±0.6)%,微生物序列占比升高至(3.1±0.1)%和(3.4±0.2)%,PRV Ct增加至25.77±0.20、25.56±0.13,PRV丰度下降至(1.6±0.3)%、(4.1±0.7)%(P<0.05)。DNaseⅠ和Benzonase使PPV Ct值增加至25.65±0.06、25.36±0.45,人类序列占比分别下降至(81.7±5.6)%、(72.8±6.7)%,微生物序列占比和PRV丰度分别上升至(11.0±4.1)、(16.1±4.7)%,(55.8±2.3)%、(39.0±8.9)%(P<0.05);RNase A处理后使PRV Ct值增加至25.20±0.11,人类序列占比降低至(85.4±5.6)%(P<0.05);溶菌酶无降低非病毒核酸效果。1%、5%、10%和20%氯仿使mtDNA和PRV Ct值增加至不低于27.17±0.21、25.68±0.04(P<0.05),10%氯仿处理后将微生�Objective To explore the influence of common methods of reducing non-viral nucleic acid on the abundance of plasma virus group.Methods Three kinds of library construction,five kinds of centrifugation conditions,two kinds of filters,four kinds of enzymes and four concentrations of chloroform were used to treat plasma samples added quantitatively 2.16 mL of pseudorabies virus(PRV)and 2.16 mL of porcine parvovirus(PPV).A total of 21.6 mL of plasma samples were processed,including 54 samples.Subsequently,nucleic acid was extracted,mitochondrial DNA(mtDNA)and two viruses were quantitated,the library of the next generation sequencing was constructed,Illumina NovaSeq 6000 was used for the next generation sequencing.The sequencing data were compared with Kraken Py 2.0 software,and the species annotation analysis was conducted.The corresponding species classification information of each segment was obtained to analyze the impact of different reducing non-viral nucleic acid methods on the relative abundance of microorganisms and two indicator viruses.Results After sequencing by Illumina NovaSeq 6000,306.27 GB raw data and 193.17 GB clean data were obtained,with Q20>90%,Q30>85%,Error Rate of 0.03%,and average GC Content of 45.02%.The DNA library construction process significantly increased the proportion of microbial sequences and the PRV abundance[(91.8±0.5)%](P<0.05);RNA library construction and combined library construction can increase the abundance of Pestivirus,an RNA virus,and the PRV abundance was(17.7±3.3)%and(8.1±1.5)%respectively.The Ct value of mtDNA was increased and the proportion of human sequence decreased to less than(89.5±1)%,while the proportion of microbial sequence increased to(2.4±0.03)%after treatment of five centrifugation conditions(P<0.05);After centrifugation at 4℃,100 g,30 min,the PRV abundance was increased to(40.6±6)%,and centrifugation at 4℃,4000 g,45 min reduced the PRV abundance to(4.1±0.01)%(P<0.05).Both of 0.22-μm filter and 0.45-μm filter increased the Ct value of mtDNA to ab

关 键 词：输血传播病毒 血浆宏病毒组 降低非病毒核 酸 高通量测序 

分 类 号：R446.5[医药卫生—诊断学] R373[医药卫生—临床医学] Q2[生物学—细胞生物学]