一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统及其构建方法
阅读说明：本技术 一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统及其构建方法 (Mycobacterium tuberculosis drug resistance detection and analysis system based on Nanopore sequencer and construction method thereof ) 是由 谷红仓 谢长校 徐振宇 王云飞 车仙荣 于 2021-06-17 设计创作，主要内容包括：本发明公开了一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统及其构建方法,属于基因检测分析领域,系统包括：微生物序列参考库模块、结核分枝杆菌耐药参考库模块、病原微生物鉴定模块、突变检测模块和耐药数据库注释模块；基于该系统的构建方法包括：构建数据比对的微生物序列参考库,将测序数据比对到微生物序列参考库并鉴定病原微生物,构建用于突变注释的结核分枝杆菌耐药参考库,将来源于结核分枝杆菌的序列比对到结核分枝杆菌参考基因组并鉴定基因突变,将结核分枝杆菌的基因突变位点与结核分枝杆菌耐药参考库关联分析突变位点的耐药性质；本系统能够快速鉴定病原微生物和检测结核分枝杆菌耐药性质,且灵敏度和特异性高。(The invention discloses a drug-resistant detection and analysis system for mycobacterium tuberculosis based on a Nanopore sequencer and a construction method thereof, belonging to the field of gene detection and analysis, wherein the system comprises: the kit comprises a microorganism sequence reference library module, a mycobacterium tuberculosis drug-resistant reference library module, a pathogenic microorganism identification module, a mutation detection module and a drug-resistant database annotation module; the construction method based on the system comprises the following steps: constructing a microbial sequence reference library for data comparison, comparing sequencing data to the microbial sequence reference library and identifying pathogenic microorganisms, constructing a mycobacterium tuberculosis drug-resistant reference library for mutation annotation, comparing a sequence derived from mycobacterium tuberculosis to a mycobacterium tuberculosis reference genome and identifying gene mutation, and correlating the gene mutation site of the mycobacterium tuberculosis with the mycobacterium tuberculosis drug-resistant reference library to analyze the drug-resistant property of the mutation site; the system can rapidly identify pathogenic microorganisms and detect the drug resistance of the mycobacterium tuberculosis, and has high sensitivity and specificity.)
技术领域
本发明涉及基因检测分析领域，特别是一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统及其构建方法。
背景技术
Nanopore测序是一种单分子、实时测序的新一代测序方法，其以单分子DNA（RNA）通过生物纳米孔的电流变化推测碱基组成而进行测序。Nanopore测序具有长度长、实时测序、按需测序以及灵活、可扩展等特点。与传统测序技术不同的是，用户可以自行掌握测序时间、地点、以及需要使用的芯片数量，这意味着用户可以根据样本的数量选择芯片数量，随数据量要求随时启动和停止实验。
结核分枝杆菌是结核病的病原体，随着抗结核药物的不断发展和居民卫生状况的改善，结核病的发病率和病死率曾大幅度下降。但自20世纪80年代以来，由于结核分枝杆菌耐药菌株，尤其是耐多药菌株的产生和传播，肺结核的发病率又有所提升，是导致人类死亡的重要疾病之一。要使肺结核发病率和病死率降低，必须实行结核病的早期诊断及其耐药性检测。采用传统的结核分枝杆菌分离培养和药敏试验通常需要1-2月时间，显然不能满足临床开展有效治疗的需要。因此，阐明结核分枝杆菌耐药机制，建立快速检测耐药结核分枝杆菌的诊断试验，已成为当前结核病研究的重要课题。
现有的结核分枝杆菌耐药检测方法耗时长，且灵敏度和特异性低，本发明解决这样的问题，具有广泛应用于临床的潜力。
发明内容
为解决现有技术的不足，本发明的目的在于提供一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统及其构建方法，本发明能够快速、准确鉴定病原微生物和检测结核分枝杆菌耐药性质。
为了达到上述目的，本发明采用如下技术方案：
一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统，包括：微生物序列参考库模块，菌株耐药参考库模块，病原微生物鉴定模块，突变检测模块和耐药数据库注释模块；
微生物序列参考库模块为通过多个数据库的微生物rRNA序列构建微生物序列参考库，对序列进行过滤，形成高可信度的微生物序列参考库；
病原微生物鉴定模块包括：测序数据样品拆分模块，数据质控处理模块，比对分析模块和物种分类模块。
测序数据样品拆分模块为：通过Guppy软件将由测序仪产生的fast5格式文件转换成fastq格式，每个样品都具有独特的barcode序列，每条序列的前一段序列就是barcode序列，将这段序列与测序仪提供的barcode序列库进行比对，将比对分数大于80的序列视为可信的分配；
数据质控处理模块为：采用NanoFilt软件进行质控，每条序列都有一个平均测序数据质量值Q，若Q值小于7的序列为不合格序列，将被删除；
比对分析模块为：将质控处理后的序列采用minimap2进行快速比对，计算reads的identity和coverage，筛选identity 大于90%且coverage大于85%的结果；
物种分类模块为：将序列与微生物序列参考库进行比对，选择比对分数最高、identity大于90%且coverage大于85%的记录作为该序列的最佳比对记录；将最佳比对记录ID与物种谱系数据库进行匹配，获取物种分类结果；
突变检测模块为对来源于菌株的reads采用minimap2比对到菌株参考基因组，采用varscan2检测基因突变位点，获得bam文件，利用samtools对bam文件进行排序，使用samtools mpileup提取mpileup文件，再使用varscan2对mpileup文件进行处理，获得突变结果并以vcf格式保存，然后使用Snpeff对突变结果进行基因注释；
菌株耐药参考库模块为以CARD数据库结合文献报道的菌株耐药位点信息整合成自建菌株耐药参考库；
耐药数据库注释模块为对基因突变位点进行注释，获取该突变位点对应的耐药药物名称。
进一步的，一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统，微生物序列参考库模块中的多个数据库包括：NCBI 16S rRNA、18S rRNA、ITS 、SILVA和UNITE数据库。
进一步的，一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统，微生物序列参考库模块中对序列进行过滤的具体方法包括：
步骤a：将来源不同的序列合并成一个综合数据库，并剔除重复序列；
步骤b：筛选有文献支持的序列作为高可信度参考序列。
进一步的，一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统，计算reads 的identity和coverage的方法为： minimap2比对结果中第13列含有前缀de:f:，该列值加上identity等于1；minimap2比对结果第2列代表序列长度，第3列代表比对其实位置，第4列代表比对终止位置，将第4列与第3列的差值除以第2列等于coverage
进一步的，一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统，菌株参考基因组为结核分枝杆菌株参考基因组，所述结核分枝杆菌株参考基因组为Mycobacterium tuberculosis H37Rv，NCBI登录号为NC_000962.3。
进一步的，一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统，自建菌株耐药参考库为自建结核分枝杆菌耐药参考库，所述自建结核分枝杆菌耐药参考库包括如下内容：gene mutation drug ahpC C-72T isoniazid ahpC C-52Tisoniazid ahpC C-81T isoniazid ahpC G-48A isoniazid embB Y319Cethambutol embB D328Y ethambutol embB F330S ethambutol embBP375A ethambutol embB D328G ethambutol embB D328Y ethambutolembB N13S ethambutol embB R213Q ethambutol embB S22Cethambutol embB N296H ethambutol embB M306L ethambutol embBM306V ethambutol embB F330V ethambutol embB G406A ethambutolembB L413P ethambutol embB D1024N ethambutol embBQ497R ethambutol embB Q497K ethambutol embB A454Tethambutol embB M306I ethambutol embB G406A ethambutol embBG406D ethambutol embB G406C ethambutol embB G406S ethambutolembB L402V ethambutol embB D354A ethambutol embB Y319Sethambutol embB S347I ethambutol embB Y334H ethambutol embBS297A ethambutol embB D299E ethambutol embB M306Lethambutol embB M306V ethambutol embB M306I ethambutol embBM306T ethambutol embB D311H ethambutol embB E504Q ethambutolembB R507G ethambutol embB D1024N ethambutol embB Y319Cethambutol embB Y333C ethambutol embB Y319N ethambutol embBT630I ethambutol embB M1000R ethambutol embB D959Aethambutol embB G745D ethambutol embB Q497K ethambutol embBQ497R ethambutol embB R471P ethambutol embB R469Pethambutol embB I465D ethambutol embB R460C ethambutol embBP446H ethambutol embB G406D ethambutol embB G406C ethambutolembB G406S ethambutol embB N399H ethambutol embB P397Qethambutol embB S380R ethambutol embB E378A ethambutolembB L239P ethambutol embB D240H ethambutol embB Y334Hethambutol embB A356V ethambutol embB A357S ethambutol embBE368A ethambutol gyrA D94Y fluoroquinolones gyrA D94Nfluoroquinolones gyrA D94H fluoroquinolones gyrA S91Pfluoroquinolones gyrA A90V fluoroquinolones gyrA D89Nfluoroquinolones gyrA D94G fluoroquinolones gyrA D94Afluoroquinolones gyrA D94N fluoroquinolones gyrA D94Gfluoroquinolones gyrA D94A fluoroquinolones gyrA D94Hfluoroquinolones gyrA D94N fluoroquinolones gyrA A90Vfluoroquinolones gyrA G88C fluoroquinolones gyrA D94Yfluoroquinolones gyrA D94G fluoroquinolones gyrA D94Yfluoroquinolones gyrA D94N fluoroquinolones gyrA P102Hfluoroquinolones gyrA T80A fluoroquinolones gyrA D94Tfluoroquinolones gyrA A74S fluoroquinolones gyrA D94Vfluoroquinolones gyrA D94G fluoroquinolones gyrA D89Vfluoroquinolones gyrA D94A fluoroquinolones gyrA D89Nfluoroquinolones gyrA D89G fluoroquinolones gyrA G88Cfluoroquinolones gyrA D94Y fluoroquinolones gyrA G88Afluoroquinolones gyrA A90V fluoroquinolones gyrA A90Gfluoroquinolones gyrA S91P fluoroquinolones gyrB E501Dfluoroquinolones gyrB N538D fluoroquinolones gyrB N510Dfluoroquinolones gyrB E540V fluoroquinolones gyrB N538Kfluoroquinolones gyrB E540D fluoroquinolones gyrB T539Pfluoroquinolones gyrB V457L fluoroquinolones gyrB V340Lfluoroquinolones gyrB D500H fluoroquinolones gyrB V457Lfluoroquinolones gyrB N499T fluoroquinolones gyrB I271Mfluoroquinolones gyrB T539N fluoroquinolones inhA C-15Tisoniazid inhA T-8C isoniazid inhA T-8A isoniazid katGR418L isoniazid katG W328F isoniazid katG W321F isoniazid katGT275V isoniazid katG M255Y isoniazid katG M255I isoniazidkatG M255C isoniazid katG W107F isoniazid katG V473Nisoniazid katG V473G isoniazid katG V473Y isoniazid katG V473Wisoniazid katG V473F isoniazid katG V473S isoniazid katGV473M isoniazid katG V473R isoniazid katG V473K isoniazid katGV473I isoniazid katG V473D isoniazid katG L472I isoniazid katGL472Q isoniazid katG L472K isoniazid katG Q471H isoniazidkatG Q471Y isoniazid katG L436G isoniazid katG T435Risoniazid katG L430V isoniazid katG L427I isoniazid katGD311S isoniazid katG D311N isoniazid katG S303C isoniazidkatG S303L isoniazid katG S315I isoniazid katG M126Iisoniazid katG S315N isoniazid katG A234G isoniazid katG V68Gisoniazid katG P131Q isoniazid katG R128Q isoniazid katG L101Pisoniazid katG R463L isoniazid katG W91R isoniazid katGP131R isoniazid katG A350S isoniazid katG Y337C isoniazidkatG W328G isoniazid katG T275P isoniazid katG T262Risoniazid katG W300G isoniazid katG Y229F isoniazid katGN138S isoniazid katG N138D isoniazid katG H108Q isoniazidkatG H108E isoniazid katG W107R isoniazid katG R104Lisoniazid katG G33V isoniazid katG G111S isoniazid katGH417Q isoniazid katG E607A isoniazid katG Q461P isoniazidkatG G125S isoniazid katG N508D isoniazid katG L147Pisoniazid katG N660D isoniazid katG A312P isoniazid katGS383P isoniazid katG H97R isoniazid katG R489S isoniazidkatG M420T isoniazid katG D387H isoniazid katG G123E isoniazidkatG G299S isoniazid katG D419H isoniazid katG M176Tisoniazid katG R632C isoniazid katG D542H isoniazid katGY64S isoniazid katG Y95C isoniazid katG P131T isoniazidkatG A139P isoniazid katG D142G isoniazid katG A162Visoniazid katG G269D isoniazid katG T306P isoniazid katGR385W isoniazid katG D387G isoniazid katG T394M isoniazid katGQ439P isoniazid katG F483L isoniazid katG A541D isoniazidkatG G32D isoniazid katG V47E isoniazid katG D194Gisoniazid katG A256T isoniazid katG T667P isoniazid katGM624V isoniazid katG A717P isoniazid katG I335T isoniazidkatG Q127P isoniazid katG Q352E isoniazid katG Y98Cisoniazid katG A379T isoniazid katG G269R isoniazid katG A431Visoniazid katG D194Y isoniazid katG S17T isoniazid katG A93Tisoniazid katG D36E isoniazid katG A110V isoniazid katGA139V isoniazid katG G300W isoniazid katG H276M isoniazid katGQ295H isoniazid katG S17N isoniazid katG G19D isoniazid katGQ224E isoniazid katG A243S isoniazid katG A550D isoniazidkatG S140N isoniazid katG G279D isoniazid katG G285Disoniazid katG G316D isoniazid katG S457I isoniazid katG G593Disoniazid katG D63E isoniazid katG N133T isoniazid katGP232S isoniazid katG Q127E isoniazid katG I317V isoniazidkatG S315R isoniazid katG S315T isoniazid katG S315Nisoniazid katG W191R isoniazid katG T667P isoniazid katG V469Lisoniazid katG R463L isoniazid katG D735N isoniazid katGG629S isoniazid katG L587M isoniazid katG S315T isoniazidkatG S315R isoniazid katG S315G isoniazid pncA D63Gpyrazinamide pncA Q10K pyrazinamide pncA T47S pyrazinamidepncA D49V pyrazinamide pncA S66P pyrazinamide pncA K96Epyrazinamide pncA A102T pyrazinamide pncA A102V pyrazinamide pncAL116R pyrazinamide pncA V125D pyrazinamide pncA I133Tpyrazinamide pncA Q141P pyrazinamide pncA R154G pyrazinamide pncAT160P pyrazinamide pncA A161P pyrazinamide pncA D12Apyrazinamide pncA D12N pyrazinamide pncA H51Q pyrazinamide pncAA171P pyrazinamide pncA H57D pyrazinamide pncA W68Lpyrazinamide pncA V139A pyrazinamide pncA R121P pyrazinamide pncAH137R pyrazinamide pncA W119R pyrazinamide pncA F94P pyrazinamidepncA T76P pyrazinamide pncA L19P pyrazinamide pncA S104Cpyrazinamide pncA I90S pyrazinamide pncA C14R pyrazinamidepncA C14Y pyrazinamide pncA G23V pyrazinamide pncA V45Gpyrazinamide pncA A46E pyrazinamide pncA D49A pyrazinamide pncAF58L pyrazinamide pncA Y68D pyrazinamide pncA S67Ppyrazinamide pncA W68R pyrazinamide pncA W68G pyrazinamide pncAW68S pyrazinamide pncA T76I pyrazinamide pncA G78Dpyrazinamide pncA L85R pyrazinamide pncA K96Q pyrazinamide pncAY103H pyrazinamide pncA Y103S pyrazinamide pncA T135Ppyrazinamide pncA D136N pyrazinamide pncA D136Y pyrazinamide pncAV139M pyrazinamide pncA A146T pyrazinamide pncA V155G pyrazinamidepncA L159R pyrazinamide pncA L172P pyrazinamide pncA V180Fpyrazinamide pncA L172A pyrazinamide pncA A171T pyrazinamidepncA H51Y pyrazinamide pncA P62H pyrazinamide pncA A171Vpyrazinamide pncA D136H pyrazinamide pncA L27P pyrazinamide pncAP54L pyrazinamide pncA C72W pyrazinamide pncA M175Vpyrazinamide pncA H59D pyrazinamide pncA A46V pyrazinamidepncA D53A pyrazinamide pncA H71E pyrazinamide pncA H82Rpyrazinamide pncA G97S pyrazinamide pncA A134V pyrazinamide pncAC138S pyrazinamide pncA T168N pyrazinamide pncA T47Ppyrazinamide pncA A121P pyrazinamide pncA Y34S pyrazinamidepncA G17D pyrazinamide pncA P54T pyrazinamide pncA A140Spyrazinamide pncA T47A pyrazinamide pncA D12E pyrazinamidepncA W68C pyrazinamide pncA T142M pyrazinamide pncA T142Kpyrazinamide pncA V139L pyrazinamide pncA C138Y pyrazinamide pncAH137P pyrazinamide pncA G132S pyrazinamide pncA N118Tpyrazinamide pncA K96N pyrazinamide pncA L85P pyrazinamidepncA C72R pyrazinamide pncA P69R pyrazinamide pncA Y41Hpyrazinamide pncA A26G pyrazinamide pncA Y34D pyrazinamide pncAD49N pyrazinamide pncA D49G pyrazinamide pncA H51Rpyrazinamide pncA P54L pyrazinamide pncA H57D pyrazinamidepncA H57R pyrazinamide pncA F58S pyrazinamide pncA P62Spyrazinamide pncA S67P pyrazinamide pncA W68R pyrazinamide pncAW68G pyrazinamide pncA P69R pyrazinamide pncA H71Rpyrazinamide pncA T76P pyrazinamide pncA H82R pyrazinamidepncA L85R pyrazinamide pncA G97S pyrazinamide pncA T100Ppyrazinamide pncA Q10P pyrazinamide pncA Q10R pyrazinamidepncA L116R pyrazinamide pncA D12A pyrazinamide pncA V128Gpyrazinamide pncA I133T pyrazinamide pncA T135P pyrazinamide pncAD136Y pyrazinamide pncA C14R pyrazinamide pncA C138R pyrazinamidepncA V139G pyrazinamide pncA Q141P pyrazinamide pncA A146Epyrazinamide pncA A146V pyrazinamide pncA L151S pyrazinamide pncAM175V pyrazinamide pncA V180F pyrazinamide pncA V180Apyrazinamide pncA V180G pyrazinamide pncA L182S pyrazinamide pncAL182S pyrazinamide pncA E174G pyrazinamide pncA L72Ppyrazinamide pncA A161G pyrazinamide pncA V157G pyrazinamide pncAR157W pyrazinamide pncA T100P pyrazinamide pncA R140G pyrazinamidepncA C138W pyrazinamide pncA K96M pyrazinamide pncA S164Ppyrazinamide pncA H57Q pyrazinamide pncA G17S pyrazinamiderpoB S531L rifampicin rpoB N518T rifampicin rpoB S522Qrifampicin rpoB K527N rifampicin rpoB R528H rifampicin rpoB S512Rrifampicin rpoB L521M rifampicin rpoB H526G rifampicin rpoB F505Lrifampicin rpoB S574L rifampicin rpoB G507S rifampicin rpoBG507D rifampicin rpoB T508P rifampicin rpoB T508A rifampicin rpoBT508H rifampicin rpoB Q513D rifampicin rpoB Q513E rifampicin rpoBS456W rifampicin rpoB D516H rifampicin rpoB N519K rifampicin rpoBG523A rifampicin rpoB H526F rifampicin rpoB H526Q rifampicin rpoBS531F rifampicin rpoB S531C rifampicin rpoB S512T rifampicin rpoBL511R rifampicin rpoB S512I rifampicin rpoB H526N rifampicinrpoB R529Q rifampicin rpoB H526T rifampicin rpoB R528Prifampicin rpoB L524S rifampicin rpoB Q517L rifampicin rpoB S509Qrifampicin rpoB S509R rifampicin rpoB E672D rifampicin rpoB H526Erifampicin rpoB L511V rifampicin rpoB L533P rifampicin rpoBD516V rifampicin rpoB T508N rifampicin rpoB S512N rifampicin rpoBP520T rifampicin rpoB S531W rifampicin rpoB S531G rifampicin rpoBH526D rifampicin rpoB H526S rifampicin rpoB K527Q rifampicin rpoBD516T rifampicin rpoB D516Y rifampicin rpoB M515I rifampicinrpoB M515V rifampicin rpoB L511P rifampicin rpoB I572Frifampicin rpoB S522L rifampicin rpoB H526Y rifampicin rpoB L538Rrifampicin rpoB V176F rifampicin rpoB Q438K rifampicin rpoB L533Rrifampicin rpoB D441V rifampicin rpoB D441Y rifampicin rpoB S447Qrifampicin rpoB H451Y rifampicin rpoB H451C rifampicin rpoB H451Drifampicin rpoB H451R rifampicin rpoB S456L rifampicin rpoB H526Crifampicin rpoB E250G rifampicin rpoB I1106T rifampicin rpoBQ975H rifampicin rpoB G836S rifampicin rpoB S450L rifampicin rpoBH526L rifampicin rpoB D516G rifampicin rpoB D516N rifampicin rpoBQ513K rifampicin rpoB Q513L rifampicin rpoB H835R rifampicin rpoBR827H rifampicin rpoB L731P rifampicin rpoB F503S rifampicinrpoB I480V rifampicin rpoB L452P rifampicin rpoB P45Lrifampicin rpoB S450W rifampicin rpoB P45S rifampicin rpoB H445Rrifampicin rpoB H445Y rifampicin rpoB D435V rifampicin rpoB D435Grifampicin rpoB D435Y rifampicin rpoB M434I rifampicin rpoB Q432Prifampicin rpoB L430P rifampicin rpoB S450L rifampicin rpoB H445Lrifampicin rpoB H445D rifampicin rpoB H445Y rifampicin rpoB S450Lrifampicin rpoB V146F rifampicin rpoB A381V rifampicin rpoB L521Prifampicin rpoB H526R rifampicin rpoB S522W rifampicin rpoB Q513Prifampicin rpoB Q490H rifampicin rpoB E504A rifampicin rpoB D518Hrifampicin rpoB Q513R rifampicin rpoB H526P rifampicin rpoB Q517Hrifampicin rpoB T508S rifampicin rpoB L511M rifampicin rpoB D516Krifampicin rpoB N518H rifampicin rpoB N518I rifampicin rpoB L538Prifampicin rpoB L545M rifampicin rpoB L571V rifampicin rpoB R633Crifampicin gidB N52T streptomycin gidB I55S streptomycin gidBD67H streptomycin gidB E92A streptomycin gidB L79Sstreptomycin gidB S70R streptomycin gidB G71V streptomycingidB Q127P streptomycin gidB A138E streptomycin gidB A183Tstreptomycin gidB L79F streptomycin gidB G117E streptomycingidB R47Q streptomycin gidB W45C streptomycin gidB L49Fstreptomycin gidB G164C streptomycin gidB P84C streptomycin gidBV188M streptomycin gidB A134E streptomycin gidB P75Sstreptomycin gidB V124G streptomycin gidB A183E streptomycingidB V188G streptomycin gidB G37V streptomycin gidB A200Estreptomycin gidB V139A streptomycin gidB P84L streptomycin gidBG30R streptomycin gidB H48Y streptomycin gidB L79Sstreptomycin gidB W148R streptomycin gidB W45S streptomycin。
一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统的构建方法，
步骤一，构建用于Nanopore测序数据比对的微生物序列参考库：
利用多个数据库的微生物rRNA序列，构建微生物序列参考库，对序列进行过滤得到高可信度的微生物序列参考库；
步骤二，测序待测样品，将测序reads比对到微生物序列参考库并鉴定病原微生物，病原微生物鉴定通过测序数据样品拆分、数据质控处理、比对分析和物种分类对测序数据进行分析；
测序数据样品拆分的方法具体包括如下步骤：
通过Guppy软件将由测序仪产生的fast5格式文件转换成fastq格式，找到样品的barcode序列，将这段序列与测序仪提供的barcode序列库进行比对，将比对分数大于80的序列视为可信的分配；
barcode序列指：每个样品都具有独特的barcode序列，每条序列的前一段序列就是barcode序列；
数据质控处理的方法为：采用NanoFilt软件进行质控，每条序列都有一个平均测序数据质量值Q，Q值小于7的序列为不合格序列；
比对分析的方法为：将质控处理后的序列采用minimap2进行快速比对，计算reads的identity和coverage，筛选identity 大于90%且coverage大于85%作为比对质量高的结果；
物种分类的方法为：将序列与微生物序列参考库进行比对，选择比对分数最高、identity大于90%且coverage大于85%的记录作为该序列的最佳比对记录；然后将最佳比对记录ID与物种谱系数据库进行匹配，获取物种分类结果；
步骤三，将来源于菌株的序列比对到菌株参考基因组并鉴定基因突变：
将来源于菌株样品序列采用minimap2比对到菌株参考基因组，获得bam文件；利用samtools对bam文件进行排序，使用samtools mpileup提取mpileup文件，再使用varscan2对mpileup文件进行处理，获得突变结果并以vcf格式保存，然后使用Snpeff对突变结果进行基因注释；
步骤四，以CARD数据库结合文献报道的菌株耐药位点信息整合成自建菌株耐药参考库；
步骤五，将基因突变位点与耐药数据库中的突变位点进行匹配关联，获取基因突变位点对应的耐药药物名称。
采用上述技术方案后，本发明的有益之处在于：
本发明基于Nanopore测序仪独特开发，整个工作流程的周转时间能够达到小于12小时，测序速度比其他测序仪更快；且测序reads读长更长，Nanopore测序reads读长可达到2M；
本发明能够快速、准确鉴定病原微生物和检测结核分枝杆菌耐药性质，灵敏度为95%、特异性为96%；
本发明构建微生物序列参考库以及结核分枝杆菌耐药参考库，为其他分析系统的构建提供了理论基础，具有广泛的应用前景；
本发明给检测菌株耐药性质提供应用基础，具有广泛的应用前景。
附图说明
图1为本发明构建方法的一种实施例的流程图。
具体实施方式
以下结合附图和具体实施例对本发明作具体的介绍。
一种基于Nanopore测序仪进行结核分枝杆菌耐药检测分析系统的构建方法，
步骤一，构建用于Nanopore测序数据比对的微生物序列参考库：
利用多个数据库的微生物rRNA序列，构建微生物序列参考库，对序列进行过滤得到高可信度的微生物序列参考库；
作为一种优选，微生物序列参考库模块中的多个数据库包括：NCBI 16S rRNA、18SrRNA、ITS 、SILVA和UNITE数据库。这些数据库是目前公开发表的较为全面的数据库，不易造成微生物漏检，需要说明的是：数据库的选取不受限制，在未来若出现更加全面的数据库也可以进行替换。
步骤二，测序待测样品，将测序reads比对到微生物序列参考库并鉴定病原微生物，病原微生物鉴定通过测序数据样品拆分、数据质控处理、比对分析和物种分类对测序数据进行分析；
测序数据样品拆分的方法具体包括如下步骤：
通过Guppy软件将由测序仪产生的fast5格式文件转换成fastq格式，找到样品的barcode序列，将这段序列与测序仪提供的barcode序列库进行比对，将比对分数大于80的序列视为可信的分配；
barcode序列指：每个样品都具有独特的barcode序列，每条序列的前一段序列就是barcode序列；
数据质控处理的方法为：采用NanoFilt软件进行质控，每条序列都有一个平均测序数据质量值Q，Q值小于7的序列为不合格序列；
比对分析的方法为：将质控处理后的序列采用minimap2进行快速比对，计算reads的identity和coverage，筛选identity 大于90%且coverage大于85%作为比对质量高的结果；
作为一种实施例，计算reads 的identity和coverage的方法为：minimap2比对结果中第13列含有前缀de:f:，该列值加上identity等于1；minimap2比对结果第2列代表序列长度，第3列代表比对其实位置，第4列代表比对终止位置，将第4列与第3列的差值除以第2列等于coverage。需要说明的是：这只是一种优选实施例，只要能计算reads 的identity和coverage的方法都适用于本发明。
物种分类的方法为：将序列与微生物序列参考库进行比对，选择比对分数最高、identity大于90%且coverage大于85%的记录作为该序列的最佳比对记录；然后将最佳比对记录ID与物种谱系数据库进行匹配，获取物种分类结果；
步骤三，将来源于菌株的序列比对到菌株参考基因组并鉴定基因突变：
将来源于菌株样品序列采用minimap2比对到菌株参考基因组，获得bam文件；利用samtools对bam文件进行排序，使用samtools mpileup提取mpileup文件，再使用varscan2对mpileup文件进行处理，获得突变结果并以vcf格式保存，然后使用Snpeff对突变结果进行基因注释；菌株参考基因组为结核分枝杆菌株参考基因组，结核分枝杆菌株参考基因组为Mycobacterium tuberculosis H37Rv，NCBI登录号为NC_000962.3。
步骤四，以CARD数据库结合文献报道的菌株耐药位点信息整合成自建菌株耐药参考库；
自建菌株耐药参考库为自建结核分枝杆菌耐药参考库，自建结核分枝杆菌耐药参考库包括如下内容：gene mutation drug ahpC C-72T isoniazid ahpC C-52Tisoniazid ahpC C-81T isoniazid ahpC G-48A isoniazid embBY319C ethambutol embB D328Y ethambutol embB F330S ethambutol embBP375A ethambutol embB D328G ethambutol embB D328Y ethambutolembB N13S ethambutol embB R213Q ethambutol embB S22Cethambutol embB N296H ethambutol embB M306L ethambutol embBM306V ethambutol embB F330V ethambutol embB G406A ethambutolembB L413P ethambutol embB D1024N ethambutol embBQ497R ethambutol embB Q497K ethambutol embB A454Tethambutol embB M306I ethambutol embB G406A ethambutol embBG406D ethambutol embB G406C ethambutol embB G406S ethambutolembB L402V ethambutol embB D354A ethambutol embB Y319Sethambutol embB S347I ethambutol embB Y334H ethambutolembB S297A ethambutol embB D299E ethambutol embB M306Lethambutol embB M306V ethambutol embB M306I ethambutol embBM306T ethambutol embB D311H ethambutol embB E504Q ethambutol embBR507G ethambutol embB D1024N ethambutol embB Y319Cethambutol embB Y333C ethambutol embB Y319N ethambutol embBT630I ethambutol embB M1000R ethambutol embB D959Aethambutol embB G745D ethambutol embB Q497K ethambutol embBQ497R ethambutol embB R471P ethambutol embB R469P ethambutolembB I465D ethambutol embB R460C ethambutol embB P446Hethambutol embB G406D ethambutol embB G406C ethambutol embBG406S ethambutol embB N399H ethambutol embB P397Qethambutol embB S380R ethambutol embB E378A ethambutol embBL239P ethambutol embB D240H ethambutol embB Y334H ethambutolembB A356V ethambutol embB A357S ethambutol embB E368Aethambutol gyrA D94Y fluoroquinolones gyrA D94N fluoroquinolonesgyrA D94H fluoroquinolones gyrA S91P fluoroquinolonesgyrA A90V fluoroquinolones gyrA D89N fluoroquinolones gyrAD94G fluoroquinolones gyrA D94A fluoroquinolones gyrA D94Nfluoroquinolones gyrA D94G fluoroquinolones gyrA D94Afluoroquinolones gyrA D94H fluoroquinolones gyrA D94Nfluoroquinolones gyrA A90V fluoroquinolones gyrA G88Cfluoroquinolones gyrA D94Y fluoroquinolones gyrA D94Gfluoroquinolones gyrA D94Y fluoroquinolones gyrA D94Nfluoroquinolones gyrA P102H fluoroquinolones gyrA T80Afluoroquinolones gyrA D94T fluoroquinolones gyrA A74Sfluoroquinolones gyrA D94V fluoroquinolones gyrA D94Gfluoroquinolones gyrA D89V fluoroquinolones gyrA D94Afluoroquinolones gyrA D89N fluoroquinolones gyrA D89Gfluoroquinolones gyrA G88C fluoroquinolones gyrA D94Yfluoroquinolones gyrA G88A fluoroquinolones gyrA A90Vfluoroquinolones gyrA A90G fluoroquinolones gyrA S91Pfluoroquinolones gyrB E501D fluoroquinolones gyrB N538Dfluoroquinolones gyrB N510D fluoroquinolones gyrB E540Vfluoroquinolones gyrB N538K fluoroquinolones gyrB E540Dfluoroquinolones gyrB T539P fluoroquinolones gyrB V457Lfluoroquinolones gyrB V340L fluoroquinolones gyrB D500Hfluoroquinolones gyrB V457L fluoroquinolones gyrB N499Tfluoroquinolones gyrB I271M fluoroquinolones gyrB T539Nfluoroquinolones inhA C-15T isoniazid inhA T-8C isoniazidinhA T-8A isoniazid katG R418L isoniazid katG W328Fisoniazid katG W321F isoniazid katG T275V isoniazid katGM255Y isoniazid katG M255I isoniazid katG M255C isoniazid katGW107F isoniazid katG V473N isoniazid katG V473G isoniazid katGV473Y isoniazid katG V473W isoniazid katG V473F isoniazidkatG V473S isoniazid katG V473M isoniazid katG V473Risoniazid katG V473K isoniazid katG V473I isoniazid katG V473Disoniazid katG L472I isoniazid katG L472Q isoniazid katGL472K isoniazid katG Q471H isoniazid katG Q471Y isoniazidkatG L436G isoniazid katG T435R isoniazid katG L430Visoniazid katG L427I isoniazid katG D311S isoniazid katGD311N isoniazid katG S303C isoniazid katG S303L isoniazidkatG S315I isoniazid katG M126I isoniazid katG S315Nisoniazid katG A234G isoniazid katG V68G isoniazid katGP131Q isoniazid katG R128Q isoniazid katG L101P isoniazidkatG R463L isoniazid katG W91R isoniazid katG P131Risoniazid katG A350S isoniazid katG Y337C isoniazid katGW328G isoniazid katG T275P isoniazid katG T262R isoniazidkatG W300G isoniazid katG Y229F isoniazid katG N138Sisoniazid katG N138D isoniazid katG H108Q isoniazid katGH108E isoniazid katG W107R isoniazid katG R104L isoniazidkatG G33V isoniazid katG G111S isoniazid katG H417Qisoniazid katG E607A isoniazid katG Q461P isoniazid katGG125S isoniazid katG N508D isoniazid katG L147P isoniazidkatG N660D isoniazid katG A312P isoniazid katG S383Pisoniazid katG H97R isoniazid katG R489S isoniazid katGM420T isoniazid katG D387H isoniazid katG G123E isoniazid katGG299S isoniazid katG D419H isoniazid katG M176T isoniazid katGR632C isoniazid katG D542H isoniazid katG Y64S isoniazidkatG Y95C isoniazid katG P131T isoniazid katG A139Pisoniazid katG D142G isoniazid katG A162V isoniazid katG G269Disoniazid katG T306P isoniazid katG R385W isoniazid katG D387Gisoniazid katG T394M isoniazid katG Q439P isoniazid katGF483L isoniazid katG A541D isoniazid katG G32D isoniazid katGV47E isoniazid katG D194G isoniazid katG A256T isoniazid katGT667P isoniazid katG M624V isoniazid katG A717P isoniazidkatG I335T isoniazid katG Q127P isoniazid katG Q352Eisoniazid katG Y98C isoniazid katG A379T isoniazid katGG269R isoniazid katG A431V isoniazid katG D194Y isoniazid katGS17T isoniazid katG A93T isoniazid katG D36E isoniazidkatG A110V isoniazid katG A139V isoniazid katG G300Wisoniazid katG H276M isoniazid katG Q295H isoniazid katG S17Nisoniazid katG G19D isoniazid katG Q224E isoniazid katGA243S isoniazid katG A550D isoniazid katG S140N isoniazidkatG G279D isoniazid katG G285D isoniazid katG G316D isoniazidkatG S457I isoniazid katG G593D isoniazid katG D63Eisoniazid katG N133T isoniazid katG P232S isoniazid katGQ127E isoniazid katG I317V isoniazid katG S315R isoniazid katGS315T isoniazid katG S315N isoniazid katG W191R isoniazidkatG T667P isoniazid katG V469L isoniazid katG R463Lisoniazid katG D735N isoniazid katG G629S isoniazid katG L587Misoniazid katG S315T isoniazid katG S315R isoniazid katGS315G isoniazid pncA D63G pyrazinamide pncA Q10Kpyrazinamide pncA T47S pyrazinamide pncA D49V pyrazinamide pncAS66P pyrazinamide pncA K96E pyrazinamide pncA A102Tpyrazinamide pncA A102V pyrazinamide pncA L116R pyrazinamidepncA V125D pyrazinamide pncA I133T pyrazinamide pncA Q141Ppyrazinamide pncA R154G pyrazinamide pncA T160P pyrazinamide pncAA161P pyrazinamide pncA D12A pyrazinamide pncA D12Npyrazinamide pncA H51Q pyrazinamide pncA A171P pyrazinamidepncA H57D pyrazinamide pncA W68L pyrazinamide pncA V139Apyrazinamide pncA R121P pyrazinamide pncA H137R pyrazinamide pncAW119R pyrazinamide pncA F94P pyrazinamide pncA T76Ppyrazinamide pncA L19P pyrazinamide pncA S104C pyrazinamidepncA I90S pyrazinamide pncA C14R pyrazinamide pncA C14Ypyrazinamide pncA G23V pyrazinamide pncA V45G pyrazinamidepncA A46E pyrazinamide pncA D49A pyrazinamide pncA F58Lpyrazinamide pncA Y68D pyrazinamide pncA S67P pyrazinamide pncAW68R pyrazinamide pncA W68G pyrazinamide pncA W68S pyrazinamidepncA T76I pyrazinamide pncA G78D pyrazinamide pncA L85Rpyrazinamide pncA K96Q pyrazinamide pncA Y103H pyrazinamide pncAY103S pyrazinamide pncA T135P pyrazinamide pncA D136Npyrazinamide pncA D136Y pyrazinamide pncA V139M pyrazinamide pncAA146T pyrazinamide pncA V155G pyrazinamide pncA L159Rpyrazinamide pncA L172P pyrazinamide pncA V180F pyrazinamidepncA L172A pyrazinamide pncA A171T pyrazinamide pncA H51Ypyrazinamide pncA P62H pyrazinamide pncA A171V pyrazinamide pncAD136H pyrazinamide pncA L27P pyrazinamide pncA P54Lpyrazinamide pncA C72W pyrazinamide pncA M175V pyrazinamide pncAH59D pyrazinamide pncA A46V pyrazinamide pncA D53Apyrazinamide pncA H71E pyrazinamide pncA H82R pyrazinamidepncA G97S pyrazinamide pncA A134V pyrazinamide pncA C138Spyrazinamide pncA T168N pyrazinamide pncA T47P pyrazinamide pncAA121P pyrazinamide pncA Y34S pyrazinamide pncA G17Dpyrazinamide pncA P54T pyrazinamide pncA A140S pyrazinamidepncA T47A pyrazinamide pncA D12E pyrazinamide pncA W68Cpyrazinamide pncA T142M pyrazinamide pncA T142K pyrazinamide pncAV139L pyrazinamide pncA C138Y pyrazinamide pncA H137Ppyrazinamide pncA G132S pyrazinamide pncA N118T pyrazinamidepncA K96N pyrazinamide pncA L85P pyrazinamide pncA C72Rpyrazinamide pncA P69R pyrazinamide pncA Y41H pyrazinamidepncA A26G pyrazinamide pncA Y34D pyrazinamide pncA D49Npyrazinamide pncA D49G pyrazinamide pncA H51R pyrazinamide pncAP54L pyrazinamide pncA H57D pyrazinamide pncA H57Rpyrazinamide pncA F58S pyrazinamide pncA P62S pyrazinamidepncA S67P pyrazinamide pncA W68R pyrazinamide pncA W68Gpyrazinamide pncA P69R pyrazinamide pncA H71R pyrazinamide pncAT76P pyrazinamide pncA H82R pyrazinamide pncA L85Rpyrazinamide pncA G97S pyrazinamide pncA T100P pyrazinamidepncA Q10P pyrazinamide pncA Q10R pyrazinamide pncA L116Rpyrazinamide pncA D12A pyrazinamide pncA V128G pyrazinamide pncAI133T pyrazinamide pncA T135P pyrazinamide pncA D136Ypyrazinamide pncA C14R pyrazinamide pncA C138R pyrazinamidepncA V139G pyrazinamide pncA Q141P pyrazinamide pncA A146Epyrazinamide pncA A146V pyrazinamide pncA L151S pyrazinamide pncAM175V pyrazinamide pncA V180F pyrazinamide pncA V180Apyrazinamide pncA V180G pyrazinamide pncA L182S pyrazinamide pncAL182S pyrazinamide pncA E174G pyrazinamide pncA L72Ppyrazinamide pncA A161G pyrazinamide pncA V157G pyrazinamide pncAR157W pyrazinamide pncA T100P pyrazinamide pncA R140Gpyrazinamide pncA C138W pyrazinamide pncA K96M pyrazinamide pncAS164P pyrazinamide pncA H57Q pyrazinamide pncA G17Spyrazinamide rpoB S531L rifampicin rpoB N518T rifampicin rpoBS522Q rifampicin rpoB K527N rifampicin rpoB R528H rifampicin rpoBS512R rifampicin rpoB L521M rifampicin rpoB H526G rifampicin rpoBF505L rifampicin rpoB S574L rifampicin rpoB G507S rifampicinrpoB G507D rifampicin rpoB T508P rifampicin rpoB T508Arifampicin rpoB T508H rifampicin rpoB Q513D rifampicin rpoB Q513Erifampicin rpoB S456W rifampicin rpoB D516H rifampicin rpoB N519Krifampicin rpoB G523A rifampicin rpoB H526F rifampicin rpoB H526Qrifampicin rpoB S531F rifampicin rpoB S531C rifampicin rpoB S512Trifampicin rpoB L511R rifampicin rpoB S512I rifampicin rpoB H526Nrifampicin rpoB R529Q rifampicin rpoB H526T rifampicin rpoBR528P rifampicin rpoB L524S rifampicin rpoB Q517L rifampicinrpoB S509Q rifampicin rpoB S509R rifampicin rpoB E672D rifampicinrpoB H526E rifampicin rpoB L511V rifampicin rpoB L533Prifampicin rpoB D516V rifampicin rpoB T508N rifampicin rpoB S512Nrifampicin rpoB P520T rifampicin rpoB S531W rifampicin rpoB S531Grifampicin rpoB H526D rifampicin rpoB H526S rifampicin rpoB K527Qrifampicin rpoB D516T rifampicin rpoB D516Y rifampicin rpoB M515Irifampicin rpoB M515V rifampicin rpoB L511P rifampicin rpoB I572Frifampicin rpoB S522L rifampicin rpoB H526Y rifampicin rpoB L538Rrifampicin rpoB V176F rifampicin rpoB Q438K rifampicin rpoB L533Rrifampicin rpoB D441V rifampicin rpoB D441Y rifampicin rpoB S447Qrifampicin rpoB H451Y rifampicin rpoB H451C rifampicin rpoB H451Drifampicin rpoB H451R rifampicin rpoB S456L rifampicin rpoB H526Crifampicin rpoB E250G rifampicin rpoB I1106T rifampicin rpoB Q975Hrifampicin rpoB G836S rifampicin rpoB S450L rifampicin rpoBH526L rifampicin rpoB D516G rifampicin rpoB D516N rifampicin rpoBQ513K rifampicin rpoB Q513L rifampicin rpoB H835R rifampicin rpoBR827H rifampicin rpoB L731P rifampicin rpoB F503S rifampicinrpoB I480V rifampicin rpoB L452P rifampicin rpoB P45Lrifampicin rpoB S450W rifampicin rpoB P45S rifampicin rpoB H445Rrifampicin rpoB H445Y rifampicin rpoB D435V rifampicin rpoB D435Grifampicin rpoB D435Y rifampicin rpoB M434I rifampicin rpoBQ432P rifampicin rpoB L430P rifampicin rpoB S450L rifampicinrpoB H445L rifampicin rpoB H445D rifampicin rpoB H445Y rifampicinrpoB S450L rifampicin rpoB V146F rifampicin rpoB A381Vrifampicin rpoB L521P rifampicin rpoB H526R rifampicin rpoBS522W rifampicin rpoB Q513P rifampicin rpoB Q490H rifampicin rpoBE504A rifampicin rpoB D518H rifampicin rpoB Q513R rifampicin rpoBH526P rifampicin rpoB Q517H rifampicin rpoB T508S rifampicinrpoB L511M rifampicin rpoB D516K rifampicin rpoB N518H rifampicinrpoB N518I rifampicin rpoB L538P rifampicin rpoB L545M rifampicinrpoB L571V rifampicin rpoB R633C rifampicin gidB N52Tstreptomycin gidB I55S streptomycin gidB D67H streptomycingidB E92A streptomycin gidB L79S streptomycin gidB S70Rstreptomycin gidB G71V streptomycin gidB Q127P streptomycingidB A138E streptomycin gidB A183T streptomycin gidB L79Fstreptomycin gidB G117E streptomycin gidB R47Q streptomycin gidBW45C streptomycin gidB L49F streptomycin gidB G164Cstreptomycin gidB P84C streptomycin gidB V188M streptomycingidB A134E streptomycin gidB P75S streptomycin gidBV124G streptomycin gidB A183E streptomycin gidB V188G streptomycingidB G37V streptomycin gidB A200E streptomycin gidB V139Astreptomycin gidB P84L streptomycin gidB G30R streptomycingidB H48Y streptomycin gidB L79S streptomycin gidB W148Rstreptomycin gidB W45S streptomycin。
步骤五，将基因突变位点与耐药数据库中的突变位点进行匹配关联，获取基因突变位点对应的耐药药物名称。
需要说明的是：本发明的耐药分析系统逻辑同样也适合于其他菌株的突变相关的耐药分析。
以下用本发明构建成的结核分枝杆菌耐药检测分析系统进行效果验证：
为了验证本发明的结果和优点，申请人收集了676个临床已知结核耐药信息（四个药物利福平、异烟肼、链霉素、乙胺丁醇）的样本，利用本发明进行结核耐药分析。结果发现，本发明针对4中结核药物耐药检测的灵敏度和特异性结果如下表1所示，抗结核药物临界浓度如表2所示。
表1结核耐药分析检测结果统计
rifampin利福平 isoniazid异烟肼 streptomycin链霉素 ethambutol乙胺丁醇 Total TP真阳性 61 56 26 22 165 TN真阴性 132 125 115 113 485 FP假阳性 4 6 5 3 18 FN假阴性 2 3 2 1 8 Total 199 190 148 139 676 Sensitivity=TP/(TP+FN)，灵敏度 97% 95% 93% 96% 95% Specificity=TN/(TN+FP)，特异性 97% 95% 96% 97% 96%
表2抗结核药物临界浓度
抗结核药物 MIC值范围（μg/ml） 临界浓度（μg/ml） 利福平 0.12-16 1 异烟肼 0.03-4 0.2 链霉素 0.25-32 2 乙胺丁醇 0.5-32 5
由表1可知：本结核分歧杆菌耐药检测分析系统能够快速、准确鉴定病原微生物和检测结核分枝杆菌耐药性质，总的灵敏度为95%、特异性为96%。
药物敏感性检测：采用微孔板药敏检测法测定最小抑菌浓度（MIC），将配制好的菌悬液转移100 微升至每个孔中，密封平板并在37 ℃孵育14 天后观察细菌的生长情况。MIC定义为与阳性对照相比没有明显可见细菌生长的最低浓度。
本发明构建微生物序列参考库以及结核分枝杆菌耐药参考库，为其他分析系统的构建提供了理论基础，具有广泛的应用前景；本发明给检测菌株耐药性质提供应用基础，具有广泛的应用前景。
除上述优选实施例外，本发明还有其他的实施方式，本领域技术人员可以根据本发明作出各种改变和变形，只要不脱离本发明的精神，均应属于本发明权利要求书中所定义的范围。
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