CN110656175A - 靶向测序gDNA对照品及其制备方法 - Google Patents
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Abstract
本发明公开一种靶向测序gDNA对照品，其特征在于，包含弱阳性对照、阳性对照，且包含SNP和INDEL两种突变类型，所述对照品涉及突变基因：TP53、APC、KIT、PTEN；本发明还公开一种靶向测序gDNA对照品的制备方法，包括步骤：1)检测突变基因各自的突变位点，找到对应阳性细胞系；2)提取所述阳性细胞系的二株细胞系的细胞gDNA，获得阳性细胞系gDNA样本；3)对阳性细胞系gDNA样本、阴性对照DNA样本进行扩增、建库、测序，检测各突变位点的原始突变频率；4)将所述阳性细胞系gDNA样本和阴性对照DNA样本进行DNA混合，形成所述靶向测序gDNA对照品。本发明减少测序工作量，节约了测序成本，具有适用范围广、节约成本、高效、可操作性强的优点。
Description
技术领域
本发明涉及生物测序技术领域，特别是涉及一种靶向测序gDNA对照品及其制备方法。
背景技术
目前特定Panel二代测序已大量用于靶向药物，特别是肿瘤类药物指导，现有的特定Panel二代测序过程的对照品一般用一个或者二个Coriell DNA标准品代替，阳性对照则需要自己检测已知样本获得突变频率后再将该已知阳性结果的样本作为阳性对照，目前市面上没有普遍适用的明确突变频率的对照品产品。因此制备普遍适用的有明确突变频率的靶向测序gDNA对照品工艺变得尤为重要。
发明内容
本发明的主要目的在于，提供一种普遍适用的有明确突变频率的靶向测序gDNA对照品，提高测序效率。
为达前述目的，本发明提供一种靶向测序gDNA对照品，适用于靶向二代测序，其包含弱阳性对照、阳性对照，且包含SNP和INDEL两种突变类型，所述对照品涉及突变基因：TP53、APC、KIT、PTEN；
所述TP53的突变位点为p.Val218del，其突变比例为10％，以及p.Pro33Arg，其突变比例为70％；
所述APC的突变位点为p.Asp1571Tyr，其突变比例为70％；
所述KIT的突变位点为P41fs，其突变比例为35％；
所述PTEN的突变位点为M134I，其突变比例为35％，以及R173H，其突变比例为35％。
TP53基因又称为P53，是一种抑癌基因，该基因编码一种分子量为53kDa的蛋白质。在人的基因里面，TP53是非常重要的抑癌基因，在正常细胞中低表达，在恶性肿瘤中高表达。在所有恶性肿瘤中，50％以上会出现该基因的突变。
APC基因是一种肿瘤抑制基因，涉及调节细胞增殖、迁移、粘着及染色体稳定等。APC基因编码的APC蛋白与β连环蛋白(beta-catenin，一种转录因子)形成复合物，导致β连环蛋白降解。APC基因的突变会导致一系列连锁反应，最终导致细胞分裂的加速，该突变与结肠腺瘤性息肉，结/直肠癌，食管癌，胃癌，胰腺癌，肝癌有关。
KIT：干细胞生长因子受体(c-kit receptor)，也被称为原癌基因c-kit或酪氨酸蛋白激酶kit或CD117，是一种受体酪氨酸激酶。正常情况下，KIT接收外部特定信息后活化，并传递到细胞内部，指导细胞有序地生长和增殖。一旦KIT发生异常，长期处于过度活跃状态，持续传递生长增殖的信号，赋予了造血前体细胞增殖或生存优势，从而导致细胞异常增多和肿瘤的发生。KIT基因突变与胃肠道间质瘤，肥大细胞病，急性髓性白血病有关。
PTEN基因是一种抑癌基因，已被证实在防止许多癌症发生和发展过程中发挥了不可或缺的作用。当PTEN基因丢缺失或突变，恶性细胞可以生长泛滥，促进癌症发展。
很多疾病，例如肺癌、结肠癌、胰腺癌、胃肠道间质瘤、肥大细胞病、急性髓性白血病等，靶向测序都会涉及TP53、APC、KIT、PTEN这几个基因，只要特定Panel二代测序检测项目中可报告范围至少包括了以上四个基因突变位点中任一个，即可使用本发明的靶向测序gDNA对照品。
本发明还提供一种靶向测序gDNA对照品的制备方法，包括步骤：
1)检测TP53、APC、KIT、PTEN突变基因各自的突变位点，找到含有所述突变位点的阳性细胞系；
2)提取1)中所述阳性细胞系的二株细胞系的细胞gDNA，获得阳性细胞系gDNA样本；
3)对阳性细胞系gDNA样本、阴性对照DNA样本进行扩增、建库、测序，检测各突变位点的原始突变频率；
4)将所述阳性细胞系gDNA样本和阴性对照DNA样本进行DNA混合，形成所述靶向测序gDNA对照品。
其中，步骤1)中通过NGS方法检测基因的突变位点。
其中，步骤2)中使用Qiagen核酸提取试剂盒提取阳性细胞系细胞gDNA。
其中，步骤3)、4)所述阴性DNA样本为Coriell DNA NA12878。
其中，步骤3)中使用AmpliSeq扩增试剂盒进行扩增。
其中，步骤3)中使用TruSeq DNA建库试剂盒进行建库。
其中，步骤3)中使用MiSeq测序平台进行测序。
本发明的靶向测序gDNA对照品既包含弱阳性对照又包含阳性对照，因此于实验设计上无需单独设置弱阳性对照和阳性对照；本发明对照品还包含了INDEL和SNP两种突变类型，可应对同时有INDEL和SNP阳性对照的特殊实验要求。
因此，本发明的对照品无需单独制作强阳性对照、弱阳性对照，可代替现有技术的2个甚至3个参照品，既减少了测序工作量，又节约了高昂的测序成本；具有适用范围广、节约成本、高效、可操作性强的优点。本发明用于靶向二代测序领域，后期可在基因缺陷诊断、肿瘤诊断、临床用药指导等区域发挥重要作用。
附图说明
图1为细胞株NCI-H650的gDNA样品文库片段大小检测结果图(重复1)
图2为细胞株NCI-H650的gDNA样品文库片段大小检测结果图(重复2)
图3为细胞株NCI-H650的gDNA样品文库片段大小检测结果图(重复3)
图4为细胞株RL95-2的gDNA样品文库片段大小检测结果图(重复1)
图5为细胞株RL95-2的gDNA样品文库片段大小检测结果图(重复2)
图6为细胞株RL95-2的gDNA样品文库片段大小检测结果图(重复3)
图7为阴性对照(NA12878)gDNA样品文库片段大小检测结果图
图8为与图1-7的检测同批的DNA Ladder的文库片段大小检测结果图
图9为对照品文库片段大小检测结果图(重复1)
图10为对照品文库片段大小检测结果图(重复2)
图11为对照品文库片段大小检测结果图(重复3)
图12为阴性对照(NA12878)文库片段大小检测结果图
图13为无模板对照(NTC)的文库片段大小检测结果图
图14为与图9-13的检测同批的DNA Ladder的文库片段大小检测结果图
具体实施方式
下面将对本发明的技术方案进行清楚、完整的描述，显然，所描述的实施例是本发明的一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都属于本发明保护的范围。
1.靶向测序gDNA对照品制备
1.1gDNA制备
1)根据肿瘤相关基因进行细胞株筛选，在本实例中选用NCI-H650和RL95-2，该两个细胞株具有稳定的TP53、APC、KIT、PTEN基因突变位点；
2)采用商业化试剂盒(Qiagen-51304)进行培养细胞；
3)对培养所得的细胞团进行gDNA提取，并使用Nanodrop超微量分光光度计(Thermo Fisher Scientific，Nanodrop 2000)，Qubit 3.0(Invitrogen Qubit3.0Fluorometer，SN：2321611130)对提取的gDNA进行定量，结果如表1所示。可知NCI-H650和RL95-2细胞株各自提取的gDNA样本的A260/A280值均在1.8-2.0之间，说明无杂质污染，且Qubit测量浓度值较高，说明gDNA的提取质量佳。
表1：gDNA浓度表
1.2扩增
采用Ion AmpliSeqTM Cancer Hotspot Panel v2商品化试剂盒(LifeTechnologies-4475346)和Ion AmpliSeqTM Library商品化试剂盒(Life Technologies-4480441)对上述gDNA样本和阴性DNA样本(NA12878)进行多重PCR扩增。
1.3文库构建
采用TruSeq Nano DNA High Throughput Library Prep Kit商品化试剂盒(Illumina-20015965)进行文库构建。使用Qubit 3.0对样本文库进行定量，NCI-H650的样本与RL95-2的样本各有三个重复，所得的样本文库浓度如表2所示。可知样本文库浓度都在2.5ng/μL以上，符合上机测序的要求。
表2文库浓度表
采用安捷伦Agilent 2100生物分析仪(Agilent Technologies，G2939A)对文库片段进行检测，结果如表2、图1至8所示，可知文库片段大小(见主峰片段大小)都在265bp左右，与理论片段大小一致，且主峰浓度比杂峰浓度高很多，避免了噪声数据，质检合格。
1.4测序
采用Reagent Kit v2,300cycles(PE)测序试剂盒(Illumina-MS-102-2002)配合Illumina MiSeq二代测序仪(Illumina MiSeqTM，SY-410-1003)，通过NGS方法对上述构建好的样本文库进行测序，检测其基因的突变位点，测得突变信息如表3所示，所述频率即为各基因突变位点在单个细胞株中的原始频率。
表3突变位点信息
1.5DNA混合
根据表3中各突变位点频率可知，NCI-H650、RL95-2DNA和NA12878的各突变位点检测，既含阳性也含部分阴性，每个位点的阳性DNA片段同时又可作为其他位点的阴性模板。
欲制备的对照品，各基因突变位点期望频率涵盖范围为10％-70％。本对照品设置高频率，可作为强阳性对照，约为70％；同时设置约10％、35％的弱阳性对照，以应对不同检测项目检测下限的需求，针对检测下限低于10％的检测，10％的突变位点为弱阳性对照，针对检测下限大于10％的检测，35％的突变位点即为弱阳性对照。同时考虑到特定Panel二代测序频率检测下限一般为10％到20％，因此所述对照品的各基因突变位点期望频率的最低限度为10％。
将RL95-2细胞株gDNA、NCI-H650细胞株gDNA、NA12878(阴性DNA样本)按表4所示进行DNA混合，将所对应加样体积用移液枪量取对应的物质，混合在一起，既为本发明对照品：TP53 p.Val218del位点的突变比例为10％，TP53 p.Pro33Arg位点的突变比例为70％，APCp.Asp1571Tyr位点的突变比例为70％，KIT P41fs位点的突变比例为35％，PTEN M134I和R173H位点的突变比例都为10％。
表4混合比例和配制过程表
2.对照品检验
将混合后的对照品进行扩增、建库、测序定量，详细步骤见1.2、1.3、1.4。样本为对照品样本，且有三个重复，以及阴性DNA样本(NA12878)、无模板对照(NTC，No TemplateControl)。
所得对照品文库浓度如表5所示，可知对照品浓度都在2.5ng/μL以上，符合上机测序要求；采用安捷伦Agilent 2100生物分析仪对文库片段进行检测，检测结果如图9至14所示，显示文库片段大小都在265bp左右，跟理论片段大小一致，杂峰很少，质检合格。测序所得对照品突变信息如表6，各个基因突变位点实际频率与理论频率很接近，且三次重复实验结果CV值都小于10％，实测频率与期望频率在一定误差范围内一致，说明所述靶向测序gDNA对照品性能稳定，结果可靠，可以作为后续实验的对照品/质控品。
表5对照品文库浓度
表6对照品突变信息表
综上所述，上述各实施例仅为本发明的较佳实施例而已，并不用以限定本发明的保护范围，凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，皆应包含在本发明的保护范围内。
Claims (8)
1.一种靶向测序gDNA对照品，其特征在于，包含弱阳性对照、阳性对照，且包含SNP和INDEL两种突变类型，所述对照品涉及突变基因：TP53、APC、KIT、PTEN；
所述TP53的突变位点为p.Val218del，其突变比例为10％，以及p.Pro33Arg，其突变比例为70％；
所述APC的突变位点为p.Asp1571Tyr，其突变比例为70％；
所述KIT的突变位点为P41fs，其突变比例为35％；
所述PTEN的突变位点为M134I，其突变比例为35％，以及R173H，其突变比例为35％。
2.一种靶向测序gDNA对照品的制备方法，其特征在于，包括步骤：
1)检测TP53、APC、KIT、PTEN突变基因各自的突变位点，找到含有所述突变位点的阳性细胞系；
2)提取1)中所述阳性细胞系的二株细胞系的细胞gDNA，获得阳性细胞系gDNA样本；
3)对阳性细胞系gDNA样本、阴性对照DNA样本进行扩增、建库、测序，检测各突变位点的原始突变频率；
4)将所述阳性细胞系gDNA样本和阴性对照DNA样本进行DNA混合，形成所述靶向测序gDNA对照品。
3.根据权利要求2所述的方法，其特征在于，步骤1)中通过NGS方法检测基因的突变位点。
4.根据权利要求2所述的方法，其特征在于，步骤2)中使用Qiagen核酸提取试剂盒提取阳性细胞系细胞gDNA。
5.根据权利要求2所述的方法，其特征在于，步骤3)、4)所述阴性DNA样本为CoriellDNA NA12878。
6.根据权利要求2所述的方法，其特征在于，步骤3)中使用AmpliSeq扩增试剂盒进行扩增。
7.根据权利要求2所述的方法，其特征在于，步骤3)中使用TruSeq DNA建库试剂盒进行建库。
8.根据权利要求2所述的方法，其特征在于，步骤3)中使用MiSeq测序平台进行测序。
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