CN104673892B - 开发恒河猴t细胞免疫组库的引物组、高通量测序方法及应用 - Google Patents
开发恒河猴t细胞免疫组库的引物组、高通量测序方法及应用 Download PDFInfo
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Abstract
本发明提供了一种开发恒河猴T细胞免疫组库的引物组、高通量测序方法及应用。本发明通过针对恒河猴TCR的可变区V区(variable)设置了26条上游引物序列,针对TCR的连接区(joining)J区设置了13条下游引物序列,通过多重PCR扩增出外周血或者其他免疫组织中高达107左右数量级的TCR序列,制备高通量测序文库并测序,通过生物信息学对恒河猴TCR基因免疫组库进行了全面分析。本发明提供的技术方案对构建恒河猴分析模型意义重大,有利于促进恒河猴模型在研究自体免疫系统疾病和传染病中的应用,也有利于人类对T细胞介导的疾病有更深入和广泛的认识。
Description
技术领域
本发明属于分子生物学领域,特别是涉及一种开发恒河猴T细胞免疫组库的引物组、高通量测序方法及应用。
背景技术
T细胞受体(TCR)在细胞免疫中发挥着非常重要的作用,TCR属于异源二聚体分子,在循环系统中,超过95%的TCR的属于αβ类型。在人类外周血中,有超过2×107特异的TCRα和TCRβ配对。所述的免疫组库的复杂性是由淋巴细胞的成熟过程中,染色体上三类基因片段,包括V、D、J基因重排以及三种基因片段连接处的插入缺失生成的。该关键区域也被成为CDR3,是主要的抗原识别位点,也称为互补决定区3(CDR3),它是TCR基因可变区中最多样和复杂的区域。
恒河猴(猕猴)这种动物模型被广泛用于研究许多重要的人类疾病,尤其是用于研究T淋巴细胞在发病机制中的作用。因此,该非人灵长类的TCR序列的表征是非常重要的。早在1992年,Gene Levinson et al.等,通过传统RT-PCR和克隆的方法,在隆恒河猴的外周血测到了23种TCRβ重排序列。后来,Emma E.M.Jaeg又在这类猕猴中发现了若干其他的TCRβ序列。通过序列比对,我们发现这些TCRβ在猕猴,黑猩猩和人类中的多样性和结构非常相似,具有很高的同源性。这些前期的工作,一共在恒河猴发现了超过23种Vβ基因,为后续建立更完整的TCRβ序列提供了至关重要的一个公共序列库。然而,由于传统克隆技术的低通量性质,在过去的二十年中恒河猴中发现的有关TCR序列的数目大约也就几百条,远远少于预估的107,这也成为了限制我们了解T细胞在免疫疾病中的演化过程的重要因素。
近年来,基于下一代测序这个强大的新技术在免疫系统中的应用,一个新的领域,称为免疫基因组库测序的技术诞生了。我们可以将循环血液中来自单个样品的数以百万计的TCR序列,在单次测序中全部一次性读取。这项技术,最早是在2009年,Weinstein等人,应用高通量测序,估计斑马鱼抗体库的大小,用这种方法,他们在每条鱼中检测到了1200至3700条抗体序列。之后Freeman等人,将该方法用于检测人T细胞检测,并且检测到了33664种不同的TCRβ克隆。随后由于测序方法的改进和通量的增加,Wang等在单个健康人中检测到了113290条独特的TCRβCDR3序列。运用统计模型,估计出的T细胞受体库的总多样性在106左右。运用这种免疫组库测序方法,科研人员检测到了抗体的不同过程的动态变化,并用于追踪淋巴细胞成熟过程以及在不同年龄组之间的差异。最近,科研人员用这项技术检测各种人类疾病。例如,检测各种白血病微小残留病变,他的灵敏度较传统的流式细胞分析法更灵敏,更可靠。这种方法也用于研究免疫系统癌症,传染性疾病如登革热,和自身免疫疾病特别是类风湿性关节炎。
尽管免疫组库测序技术在人类基础研究领域中已经有广泛应用,但这种技术在其他模式生物的使用非常有限,仅限于小鼠和斑马鱼。猕猴是一种重要的动物模型,尤其是对人类疾病的研究。超过70种传染病的研究都在恒河猴模型中进行,特别是由人类免疫缺陷病毒(HIV)引起的获得性免疫缺陷综合症 (AIDS),结核病(TB)和疟疾。因此,建立对恒河猴TCRβ进行大规模测序的方案对构建该物种分析模型十分必要。
发明内容
鉴于此,本发明提供了一种开发恒河猴T细胞免疫组库的引物组、高通量测序方法及应用。
第一方面,本发明提供了一种开发恒河猴T细胞免疫组库的引物组,包括26条上游引物和13条下游引物,其中,所述26条上游引物为如SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列,所述13条上游引物为如SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列。
本发明通过针对恒河猴TCR的可变区V区(variable)设置至少26条上游引物序列,针对TCR的连接区(joining)J区设置至少13条下游引物序列,通过多重PCR扩增出目的链的PCR产物,获得高通量测序文库。
本发明采用至少13条下游引物序列与所述至少26条上游引物序列随机组合成配对引物,然后进行多重PCR反应,扩增TCRβ区。
本发明针对TCR所有的64类V和13类J基因家族进行了比对分析,26条上游引物序列针对TCR的CDR3区上游(即FR3区),13条下游引物针对J基因,扩增TCR的CDR3区。
优选地,所述的上游引物为3’端比SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列多或少1~3个碱基的核苷酸序列,所述下游引物的为3’端比SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列多或少1~3个碱基的核苷酸序列;
所述上游引物或下游引物多出的1~3个碱基为与目的TCR互补的碱基。
本发明所述的与设计目的TCR互补的碱基是指:针对恒河猴TCR的可变区V区(variable)或针对TCR的连接区(joining)J区设计引物。
第二方面,本发明提供了一种检测恒河猴T细胞免疫组库的高通量测序方法,包括如下步骤:
提取恒河猴基因组DNA或总RNA;
采用开发恒河猴T细胞免疫组库的引物组,进行多重PCR反应;
将所得多重PCR产物进行高通量测序;
其中,所述开发恒河猴T细胞免疫组库的引物组包括26条上游引物和13条下游引物,其中,所述26条上游引物为如SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列,所述13条上游引物为如SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列。
优选地,所述多重PCR反应的体系中,26条上游引物等摩尔混合;13条下游引物等摩尔混合。
优选地,所述多重PCR反应的体系中,模板量为1~3ug/50ul体系。
优选地,所述多重PCR反应程序为:
优选地,所述多重PCR反应结束后,电泳,割胶回收片段长度为100-150bp的DNA片段。
优选地,所述的上游引物为3’端比SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列多或少1~3个碱基的核苷酸序列,所述下游引物的为3’端比SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列多或少1~3个碱基的核苷酸序列;
所述上游引物或下游引物多出的1~3个碱基为与目的TCR互补的碱基。
本发明提供的TCR高通量测序文库能获得的长度和数量丰富的TCR序列,有利于TCR序列的多态性程度分析及TCRCDR3区长度多态性分布分析。
优选地,所述T淋巴细胞受体(TCR)待测序列为采用RNA试剂盒提取人外周血单个核细胞的总RNA或者获得的基因组DNA。
总RNA样品需要先进行逆转录,再进行多重PCR。
第三方面,本发明提供了一种构建恒河猴T细胞免疫组库的方法,包括:
提取恒河猴基因组DNA或总RNA;
采用开发恒河猴T细胞免疫组库的引物组,进行多重PCR反应;
将所得多重PCR产物进行高通量测序;
生物信息学分析,对高通量测序结果进行定量分析;
其中,所述开发恒河猴T细胞免疫组库的引物组包括26条上游引物和13条下游引物,其中,所述26条上游引物为如SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列,所述13条上游引物为如SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列。
在高通量测序平台的基础上,通过对恒河猴TCR基因免疫组库进行全面的生物信息学分析,获得了TCR在VDJ重组时的基因偏好性(usage patterns),基因组合、连接多样性信息,以及CDR3序列中氨基酸的偏好性(usage patterns)、CDR3氨基酸序列的长度多样性以及连接处N端碱基的特性。正是这些因素形成数量庞大且种类多样的TCR受体库。
优选地,所述生物信息学分析采用了发明人自编的在线生物学分析软件“iRAP”,网址为: http://sustc-genome.org.cn/irap。
优选地,所述多重PCR反应的体系中,26条上游引物等摩尔混合;13条下游引物等摩尔混合。
优选地,所述的上游引物为3’端比SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列多或少1~3个碱基的核苷酸序列,所述下游引物的为3’端比SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列多或少1~3个碱基的核苷酸序列;
所述上游引物或下游引物多出的1~3个碱基为与目的TCR互补的碱基。
优选地,所述多重PCR反应的体系中,模板量为1~3ug/50ul体系。
优选地,所述多重PCR反应程序为:
优选地,所述多重PCR反应结束后,电泳,割胶回收片段长度为100-150bp的DNA片段。
第四方面,本发明提供了一种如第一方面所述的开发恒河猴T细胞免疫组库的引物组在检测恒河猴T细胞受体、对恒河猴T细胞受体进行高通量测序、构建恒河猴T细胞免疫组库、以及制备恒河猴T细胞免疫组库试剂盒中的应用。
本发明提供的技术方案对构建恒河猴分析模型意义重大,有利于促进恒河猴模型在研究自体免疫系统疾病和传染病中的应用,也有利于人类对T细胞介导的疾病有更深入和广泛的认识。
采用本发明的技术方案,达到如下有益效果:
1)获得了恒河猴1.26(百万)条TCR序列;
2)获得了恒河猴643,570条特异性TCR克隆;
2)获得了恒河猴270,557条特异的TCR CDR3序列,这大大扩展了对该物种免疫TCR的认识。而目前IMGT和NCBI数据库所有恒河猴TCR相关的序列只有几百条。
附图说明
图1-a为TCRβVDJ重组、TCR文库构建以及生物信息流程示意图;
图1-b为高通量测序序列的统计分析结果;
图1-c为高通量测序TCRβ序列的频率直方图;
图2-a TCRβV基因使用频率分布
图2-b TCRβJ基因使用频率分布
图2-c TCRβD基因使用频率分布
图2-d为整个VDJ基因组的3D分布图;
图2-e为不同VDJ重组频率和的数量对应分布情况;
图2-f为测序深度和VDJ重组片段饱和度分析结果;
图3-a TCRβ的CDR3氨基酸长度分布和不同J基因使用分布;
图3-b为12aa长度的TCRβ的CDR3氨基酸组成成份分析;
图3-c为TCRβ的CDR3序列中,不同氨基酸采用的密码子情况;
图3-d为不同TCRβ的CDR3序列的出现频率图;
图3-e为CDR3序列组大小和多样性统计分析;
图4-a为VDJ重组片段区域的13个分区;
图4-b为VDJ重组片段区域中,插入或缺失的片段长度分布情况;
图5-a为PCR偏差的分析;
图5-b为测序误差的评估。
具体实施方式
图1-a为TCRβVDJ重组、TCR文库构建以及生物信息流程示意图;图1-a显示TCRβ的多样性来源 于VDJ重组以及连接多样性,我们通过设计多重PCR引物组扩增TCR基因,并进行测序和分析;根据图1,本发明提供了如下实施例:
材料及试剂说明:
恒河猴:10年大的母猴,来源于广州生物医药与健康研究院(GIBH),饲养按行业标准。非特殊说明,本发明实施例采用的试剂均为市售商品,本发明实施例采用的数据库均为公开的在线数据库。
实施例1
T淋巴细胞受体(TCR)基因组DNA样品的制备,包括如下步骤:
(1)收集的新鲜外周血样本各10毫升(ml),按LymphoPrep试剂盒(Axis-shield,Cat.No.AS1114544UK)说明书操作,获得相对较纯的PBMC;
(2)采用PureLink Genomic DNA Mini Kit(Life Technology,Cat.No:K1820-00)试剂盒提取步骤(1)所得细胞的基因组DNA,并用Nanodrop2000(Thermo)测定DNA的浓度及纯度,然后保存基因组DNA。
实施例2
采用开发恒河猴T细胞免疫组库的引物组进行多重PCR,构建TCRβ高通量测序文库,进行高通量测序,包括如下步骤:
(1)以实施例1所得基因组DNA为扩增模板,取TCR引物,再采用QIAGEN公司Multiplex PCR试剂盒(货号:206143),按试剂盒说明书配置多重PCR体系,其中,每个前向引物等摩尔混合,引物总浓度是10微摩尔,每个反向引物等摩尔混合,引物总浓度是10微摩尔,模板量可以调整,本实施例中采用3ug,1ug也可以做出来,但是多样性会下降。
该体系中的上游引物为如SEQ ID NO:1~SEQ ID NO:26所示引物的等摩尔混合物;下游引物为如SEQ ID NO:27~SEQ ID NO:39所示引物的等摩尔混合物。
(2)多重PCR
为方便送测,若无特别说明,本发明实施例进行多重PCR时,分别在上游引物和下游引物加上测序接头,具体为:在如SEQ ID NO:1~SEQ ID NO:26所示的引物的5’端分别接上illumina测序公司的上游引物接头序列,在下游引物的5’端分别接上illumina测序公司的下游引物接头序列,具体步骤参照illumina高通量测序文库构建说明书;
再按下述多重PCR的条件设置PCR仪器程序,进行多重PCR:
72℃7min,PCR结束后,4℃保存PCR产物并电泳检测,挑选片段长度约为120bp的TCRβ片段,割胶回收,得到纯化后的TCRβ片段,胶回收步骤采用QIAGEN公司QIAquick胶纯化试剂盒,按常规实验室操作进行;Nanodrop 2000测试DNA浓度,并送公司进行高通量测序(采用Illumina hiseq2000测序,2*100pair-end)。
本发明实施例设计26条上游引物,13条下游引物对TCRβ免疫组重链进行分析:
1)设计引物:针对TCR所有的64类V和13类J基因进行了比对分析,采用Oligo 7.0和MFEprimer-2.0对引物二聚体以及茎环错配进行分析,在TCR的CDR3区上游(即FR3区)设置了上游引物,针对J基因下游设计反向引物,扩增TCRβ基因。
本实施例提供的引物组覆盖了大部分TCRβVDJ重组片段。由于很小的序列变化将导致引物扩增效果显著降低,本发明选取了扩增效果最佳的引物组。
2)多重PCR产物大小在120bp左右。
3)采用Illumina Hiseq 2*100pair-end测序。
4)对高通量测序结果的生物信息学分析采用本发明人自研在线软件ImmuneRepertoire Analysis Pipeline(iRAP,http://www.sustc-genome.org.cn/irap/)
生物信息学分析结果如下:
一、TCR基因序列的分析
采用本发明的引物组以及多重PCR构库后,高通量测序得到大约1.69(百万)条序列。经过和TCRβ比对分析,得到图1-b所示结果,图1-b为高通量测序序列的统计分析结果,由图1-b可知,1.69(百万)条序列中,1.26(百万)条为TCRβ序列,其中,1.07(百万)条为可正常编码氨基酸序列的CDR3序列,另有0.19(百万)条在CDR3不能正常编码序列,T细胞中含有的out-of-frame序列,如果不被nonsense-mediated decay(NMD)途径沉默或破坏,将会引起严重疾病。
CDR3区域是TCRβ基因中多样性最丰富的区域,是抗原结合位点的关键,本次分析,我们发现了0.23(百万)条CDR3氨基酸序列,这可以和人类CDR3多样性相匹敌。图1-c为高通量测序TCRβ序列的频率直方图,结果显示,25.2%的TCR序列只有1个拷贝,所有TCR CDR3的寡克隆指数(oligoclonality index,OI)为0.63,表明CDR3序列拷贝数的分布非常不一致,变化幅度很大。
二、VDJ基因出现频率的规律
根据IMGT数据库可知,恒河猴具有64种V,2种D和13种J基因片段。本实施例参考现有数据库,通过比对分析每一条高通量测序所得TCRβ序列,获得57种V,2种D和13种J基因片段,由此可知,本发明提供的多重PCR引物组覆盖了大部分VDJ基因片段。
根据分析,V、D、J基因片段的丰度非常不一致,有些片段丰度显著高于其他片段,结果如图2-a~c所示。图2-a TCRβV基因使用频率分布,图2-b TCRβJ基因使用频率分布,图2-c TCRβD基因使用频率分布。横坐标为不同的V,D,J基因家族,不同的V基因家族,纵坐标为每类V基因家族的序列占 总序列数(Total reads)的百分比,可以看出:丰度最高的V基因片段为V6-1,V6-3和V6-4,丰度排前8的V基因片段共占所有V基因的50%。同样,J基因中,J2-7占所有J基因的27%。此外,我们发现,D1的丰度高于D2。图2-a~c的现象可能是受重组过程中染色体空间位置影响,即空间位置越近的越容易重组在一起。
由上可知,恒河猴具有1664中可能的VDJ重组方式(64V×2D×13J=1,664VDJ),本发明实施例获得了1094中VDJ重组,覆盖率达66%,结果如图2-d所示。
图2-d为整个VDJ基因组的3D分布图,3个坐标轴代表了所有可能的VDJ值,每一个(V,D,J)坐标代表一个特异的VDJ重组方式,图中小球的体积代表了该点对应的VDJ重组方式的数量。红、绿、蓝(竖轴从上至下)分别代表D2、D1和未确定的D,由于TCR D是较复杂的的区域,可能存在不稳定的缺失或转化,导致在VDJ重组过程中的D基因缺失,与此相符的,本发明分析发现,在对D基因的测序结果进行分析后,大概有16.8%的D基因测序结果比对不到确定的、对应的D基因,这些基因归类为未确定的D。
由于VDJ3种基因片段丰度的不同,我们预测VDJ重组种类也有所差异,与此相符的是,如图2-d中,在所有1094种重复方式中,不同VDJ重组方式的丰度在0.01%-1.85%之间变化,丰度最高的VDJ重组(V10-1/D2/J2-7)具有12057条测序所得序列,然而,最小丰度的VDJ重组至对应1条测序所得序列。
进一步地,如图2-e所示,图2-e为不同VDJ重组频率和数量对应分布情况。发明人随机在测序所得基因文库中选择一定比例的基因片段,统计了不同VDJ重组种类的数量。由图2-e可知,VDJ重组种类丰度变化曲线具有长尾巴分布规律。
图2-f为为测序深度和VDJ重组片段饱和度分析结果。
通过多重采样分析结果显示目前的测序深度对于TCR的多样性已经接近饱和(Fig.2f).
三、CDR3的特点
CDR3氨基酸序列中,1个氨基酸(aa)的变化可能导致受体构象的改变,因此,CDR3氨基酸序列长度的变化可以反映出CDR3基因连接区的多样性。本发明实施例中,恒河猴CDR3的定义采用人类中一样的定义,即,从TRBV最后一个半胱氨酸到TRBJ片段的FGXG结构域中的苯丙氨酸之间的序列。可以发现,CDR3的aa长度为10-16(核苷酸序列30-48bp),其中,84%的CDR3序列长度为11-13aa之间,如图3-a所示。图3-a为TCRβ的CDR3氨基酸长度分布和不同J基因使用分布;横坐标为氨基酸序列的长度,纵坐标为某种长度CDR3序列的数量,其中,柱子中的不同颜色代表了不同的J基因,由图3-a可知,所有的TCRβ的CDR3氨基酸序列均采用了J2-1基因;此外,12个aa长度的TCRβ的CDR3序列数量最庞大;图3-a还显示了部分具有13个氨基酸(aa)的序列的数量。
我们以12aa长度的TCRβ的CDR3序列为目标,采用Crooks et al.中的WebLogo法,研究CDR3氨基酸组成组成成份,如图3-b所示。图3-b为12aa长度的TCRβ的CDR3序列的组成情况。
图3-c反应了TCRβ的CDR3区域中氨基酸的种类,以及不同氨基酸采用的密码子情况;
图3-d为不同TCRβCDR3序列的丰度图;由图3-d可知,多数CDR3序列少于10个拷贝。
图3-e为CDR3序列组大小和多样性统计分析。
该统计是用于分析我们的测序深度是否足够用于评估目前的TCR多样性,结果显示我们的测序深度已经接近饱和,足够用于评估。
四、恒河猴的总TCRβCDR3多样性分析
CDR3多样性是正常免疫反应的基础。现有研究表明人类TCR□中特异的CDR3有340,000种。为了确定我们的测序是否覆盖恒河猴主要的特异性CDR3种类,我们参照FisherRA et al.中提到的采样-重采样统计分析方法对样本的大小和多样性进行了分析。结果如图3-e所示,相比由该方法预测的262,412种特异性CDR3序列,我们实际获得的特异性CDR3序列为235,573中,覆盖了总特异性CDR3序列的90%。
五、恒河猴连接区的多样性分析
恒河猴TCR多样性不仅来源于VDJ重组,很多TCR还来源于CDR3区域V-D和D-J之间连接区核苷酸的插入或缺失,此类插入或缺失不依赖于模板,而是通过末端脱氧核苷酸转移酶实现,在本发明中,我们参照IMGT数据库的定义,将恒河猴的TCRβ的连接区划分为13个区域,如图4-a所示。图4-a为VDJ重组片段区域的13个分区;我们对所有270,557种CDR3的核苷酸序列的每个区域均进行研究,分析了不同插入或缺失序列的长度分布情况,如图4-b所示。图4-b为VDJ重组片段区域的13个分区中,插入或缺失的片段长度分布情况,x轴表示13种片段,y轴表示VDJ重组过程中,插入或缺失的片段长度,z轴表示某种特定片段长度所占比例。z轴正方形为插入,负方向为缺失。N1和N2插入片段的平均长度分别为4bp和3bp。
注:
本发明测序结果分析方法:Illumina Hiseq 2000(paired-end 2*100)测序得到2,645,432条序列,我们用FLASH软件拼接有重叠的paired-end序列。拼接后的结果采用IMGT-High V-QUEST比对V、D和J基因。同源性低的从测序所得文库中去除。CDR3区域的起点和重点、编码框以及丰度的定义来源于IMGT。
我们用采样-重采样的方式研究整个CDR3免疫组库的大小,第一次取样中,占总CDR3免疫组库1% 的序列中,获得953条特异性的CDR3序列,第二次取样中,占总CDR3免疫组库1%的序列中,获得885条特异性的CDR3序列,第三次取样中,占总CDR3免疫组库1%的序列中,获得9831条特异性的CDR3序列,我们总结出对数衰减值(R2=0.97)。所有样品计算后,将所有特异性CDR3序列求和,可预测出整个CDR3免疫组库的大小。
我们用寡克隆指数(oligoclonality index,OI,见式1)来评估CDR3序列丰度的分布,如果是单克隆则OI值为1,如果不止1个克隆,OI值记为0,式1的定义基于Gini相关因素:
其中,S代表所有的TCRβCDR3克隆
Si表示特定TCRβCDR3克隆的丰度
测序所得的TCRβCDR3克隆
表示TCRβCDR3克隆的相对丰度
表示递减顺序下,TCRβCDR3克隆的累加相对丰度。
实施例3
矫正多重PCR引物扩增误差的方法,包括:
本发明的目的之一是分析TCR序列的丰度情况,然而,PCR扩增过程中,由于PCR引物对不同基因片段的扩增偏好,会影响TCR序列丰度的真实情况。
为了评估这类系统误差,我们采用了Robins et al中的方法,我们按照实施例1的方法提取1只恒河猴的基因组,均分为6管,3管采用25循环的多重PCR(参照实施例2),3管采用30个循环,每管扩增产物均由Illumina Miseq测序,在25循环的实验组中,共发现10,345条特异的TCR CDR3序列,15.3%were also found in the 30-cycle PCR experiment.
我们将25循环组中发现的19658条测序结果和30循环组中发现的32647条测序结果进行分析,发现了如图5-a所示的线性关系。图5-a为PCR偏差的分析。对比25循环组和30循环组,假设误差全部来源于PCR误差,则平均每个PCR循环引入1.033,因此,整个30循环组引入的误差为1.03330=2.7。
矫正高通量测序误差的方法,包括:
(1)制备多重PCR模板
(1a)构建质粒文库
参照实施例1和实施例2步骤(1)-(2),进行多重PCR;
采用Invitrogen公司TOPO克隆试剂盒将实施例2步骤(2)胶回收纯化后的PCR片段与TA克隆的质粒载体连接;然后取2-3μl连接产物,加入至100μl JM109感受态细胞中,轻轻混匀,冰中静置30分钟后,再在42℃水浴加热60秒钟,然后迅速置于冰上1-2分钟;往转化产物中加入890μlLB培养基(amp-),37℃、175rpm振荡培养60分钟,然后将培养液以4000rmp离心1min,弃上清若干,剩下的菌液混匀后取3/4的量在含有X-Gal(10ul)、IPTG(100ng20ul)、Amp的L-琼脂平板培养基上涂布,待菌液完全被培养基吸收后,37度倒置培养12-16h,形成单菌落,计数白色、蓝色菌落;
挑取每个平板上单个白色菌落,加到600ul LB培养液(Amp+),恒温孵育箱225rpm37度震荡培养2-5h,然后取2.5ul菌液做PCR(25ul体系);
将得到的PCR产物进行琼脂糖凝胶电泳,确认载体中插入片段的长度大小,测序后选取阳性克隆组成TCR质粒文库;
(1b)挑选26种质粒混合制备多重PCR模板
在(1a)所得TCR质粒文库中挑选出26种质粒,挑选标准为:根据BCR的C区、V、D和J区的特征,所得克隆与IMGT数据库中的序列进行生物信息学分析并分类,26种质粒为26种V区beta基因质粒,序列已知。
将该26种TCR质粒等摩尔混合后,作为多重PCR模板,每种质粒的添加数量是已知的,即每条DNA的数量是已知的;
(2)设置多重PCR引物
多重PCR引物为实施例2步骤1中多重PCR引物;
(3)多重PCR
取步骤(1)制备的多重PCR模板与多重PCR引物,参照实施例2步骤2的方法进行多重PCR,不同点在于设置3个实验组,每组平行3管,PCR循环次数分别设置为20、25、30,分别得到的PCR产物,将得到的PCR产物进行2%琼脂糖凝胶电泳,确认载体中插入片段的长度大小100-150bp,胶回收(Qiagen,Cat.No.28704,Valencia,CA);
(4)测序分析
将所有步骤3的PCR产物送测(Illumina Miseq 2*150),测序结果和原始质粒比对,计算误差(方法参照Robins et el.)。结果如图5-b所示。图5-b为测序误差的评价结果。
由图5-b可知,95.1%的测序结果和原始质粒完全一致,4.9%的测序结果在CDR3区域有至少1个bp的错误,经过计算,得到CDR3的测序误差为0.0186%/bp。
注:
本发明人采用的引物组:
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Claims (8)
1.一种开发恒河猴T细胞免疫组库的引物组,其特征在于,包括26条上游引物和13条下游引物,其中,所述26条上游引物为如SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列,所述13条下游引物为如SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列。
2.一种检测恒河猴T细胞免疫组库的高通量测序方法,其特征在于,包括如下步骤:
提取恒河猴基因组DNA或总RNA;
采用开发恒河猴T细胞免疫组库的引物组,进行多重PCR反应;
将所得多重PCR产物进行高通量测序;
其中,所述开发恒河猴T细胞免疫组库的引物组包括26条上游引物和13条下游引物,其中,所述26条上游引物为如SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列,所述13条下游引物为如SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列。
3.如权利要求2所述的检测恒河猴T细胞免疫组库的高通量测序方法,其特征在于,所述多重PCR反应的体系中,26条上游引物等摩尔混合;13条下游引物等摩尔混合。
4.如权利要求2所述的检测恒河猴T细胞免疫组库的高通量测序方法,其特征在于,所述多重PCR反应的体系中,模板量为1~3ug/50ul体系。
5.如权利要求2所述的检测恒河猴T细胞免疫组库的高通量测序方法,其特征在于,所述多重PCR反应程序为:
6.如权利要求2所述的检测恒河猴T细胞免疫组库的高通量测序方法,其特征在于,所述多重PCR反应结束后,电泳,割胶回收片段长度为100-150bp的DNA片段。
7.一种构建恒河猴T细胞免疫组库的方法,其特征在于,包括:
提取恒河猴基因组DNA或总RNA;
采用开发恒河猴T细胞免疫组库的引物组,进行多重PCR反应;
将所得多重PCR产物进行高通量测序;
生物信息学分析,对高通量测序结果进行定量分析;
其中,所述开发恒河猴T细胞免疫组库的引物组包括26条上游引物和13条下游引物,其中,所述26条上游引物为如SEQ ID NO:1~SEQ ID NO:26所示的核苷酸序列,所述13条下游引物为如SEQ ID NO:27~SEQ ID NO:39所示的核苷酸序列。
8.一种如权利要求1所述的开发恒河猴T细胞免疫组库的引物组在检测恒河猴T细胞受体、对恒河猴T细胞受体进行高通量测序、构建恒河猴T细胞免疫组库、以及制备构建恒河猴T细胞免疫组库的试剂盒中的应用。
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