CN112708676A - 一种用于dna同源重组修复基因检测的标准品及其制备方法 - Google Patents
一种用于dna同源重组修复基因检测的标准品及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种用于DNA同源重组修复基因检测的标准品及其制备方法和应用,所述标准品包括基因panel;所述基因panel包括28种基因,所述28种基因对应的名称如下:BRCA1、BRCA2、ATM、ATR、BARD1、BLM、BRIP1、CDK12、CHEK1、CHEK2、FANCA、FANCC、FANCD2、FANCF、FANCI、FANCL、FANCM、MRE11A、NBN、PALB2、PPP2R2A、RAD50、RAD51B、RAD51C、RAD51D、RAD52、RAD54L和RPA1。本发明中通过对特定的肿瘤细胞株做NGS检测,筛选得到与HRR相关的28种基因,并通过特定比例混合,使得每种基因都包含多样的突变类型以及突变频率全覆盖,再进过精确定量后,形成标准品,进而能够用于HRR检测中的性能评价。
Description
技术领域
本发明涉及生物医药技术领域,具体涉及一种用于DNA同源重组修复基因检测的标准品及其制备方法。
背景技术
乳腺癌(breast cancer,BC)已成为中国女性最常见的恶性肿瘤,发病率逐年攀升。随着全基因组关联性研究(genomewide association studies,GWAS)的进展,一系列与BC发生发展密切相关的DNA损伤修复相关基因突变与单核苷酸多态性(single nucleotidepolymorphisms,SNP)位点得以发现。以BRCA1/2为核心的肿瘤抑制因子网络主要参与包括电离辐射等多种因素导致的DNA双链断裂(doublestrands breaks,DSB)的修复过程,通过与多种蛋白组成功能复合体调控同源重组修复(homologous recombination repair,HRR),此网络中关键因子突变或SNP可能造成DNA修复能力缺失,引起基因组失稳,导致癌症发生。
同时最新的NCCN指南在前列腺癌中,也明确了HRR和前列腺癌的相关性,要求对于初诊未进行风险评估、极低风险至中风险的前列腺癌患者,其家族史的获得及遗传咨询是检测前的必要步骤:对于具有明确相关家族史、已知家族成员携带胚系致病基因突变的上述风险级别患者,推荐进行DNA损伤修复相关基因(特别是BRCA2、BRCA1、ATM、PALB2、CHEK2、MLH1、MSH2、MSH6、PMS2)的胚系变异检测;对于家族史不详的上述风险级别患者,需要结合临床特征进行遗传咨询后综合判断是否有必要进行相关检测。对于高风险、极高风险、局部进展及转移性前列腺癌患者,推荐进行DNA修复基因(特别是BRCA2、BRCA1、ATM、PALB2、CHEK2、MLH1、MSH2、MSH6、PMS2)的胚系变异检测。
另外在其他肿瘤中,比如胰腺癌、卵巢癌、膀胱癌等等,尤其是卵巢癌,发现DNA交联剂(如铂类药物)以及聚ADP核糖聚合酶抑制剂(Poly ADP-ribose Polymeraseinhibitors,PARPi)在这些肿瘤中,当出现HRR异常导致的HRD时,病人是明确能获益的。
DNA同源重组修复(Homologous Recombination Repair,HRR)是DNA双链损伤的重要修复方式。HRR是一条涉及到多个步骤的复杂的信号通路,其中关键蛋白为BRCA1和BRCA2。根据ARIEL3的报告,通路主要涉及BRCA1,BRCA2,ATM,ATR,BARD1,BLM,BRIP1,CDK12,CHEK1,CHEK2,FANCA,FANCC,FANCD2,FANCF,FANCI,FANCL,FANCM,MRE11A,NBN,PALB2,PPP2R2A,RAD50,RAD51B,RAD51C,RAD51D,RAD52,RAD54L和RPA1这28种基因。这些基因的很多突变都能引起DNA修复的异常,与肿瘤的发生、诊断、治疗息息相关。
围绕HRR相关基因开发的检测试剂盒、检测方法很多,有Q-PCR,AMRAS-PCR,NGS等等,这些方法从原理上都能对HRR相关基因做出定性、定量的判断,进而能分析临床的意义,但是对于任何一种方法的质控,方法的性能评价,检测限怎么样?重复性怎么样?准确性怎么样?市场上并没有一个比较理想的标准品来回答这些问题。
主要原因是HRR相关的基因个数比较多,而且每个基因都不存在热点突变,也就是说但凡是Pathogenic,或者Likely Pathogenic,甚至是Uncertain Significance,都是有可能导致基因的异常,进而需要被关注,需要被检测的。遵循这个逻辑的话,以往质粒标准品的做法肯定是不合适的,一来质粒是纯品,不是全基因组,与临床样本差异巨大,二来也无法全部设计你想要这么多基因的热点突变,在第三类医疗器械的IVD审评指导原则上也明确建议不能使用质粒标准品。
因此开发一种“与临床背景类似的、可重复生产的、含有多个基因的多个突变、含有不同临床意义的突变、含有不同突变类型的位点”的检测标准品,是市场亟待需求的,能满足市场对HRR检测的质控、性能评价的需求。
发明内容
本发明的目的在于提供一种用于DNA同源重组修复基因检测的标准品及其制备方法,该标准品含有HRR相关的28种基因,且每种基因都包含多样的突变类型以及突变频率全覆盖,能够用于HRR检测中的质控。
为了实现本发明的上述目的,特采用以下技术方案:
本发明第一方面提供一种用于DNA同源重组修复基因检测的标准品,所述标准品包括基因panel;
所述基因panel包括28种基因,所述28种基因对应的名称如下:
BRCA1、BRCA2、ATM、ATR、BARD1、BLM、BRIP1、CDK12、CHEK1、CHEK2、FANCA、FANCC、FANCD2、FANCF、FANCI、FANCL、FANCM、MRE11A、NBN、PALB2、PPP2R2A、RAD50、RAD51B、RAD51C、RAD51D、RAD52、RAD54L和RPA1。
优选地,所述基因panel来源于肿瘤细胞株,所述肿瘤细胞株包括HCC1143、HCC1599和HCC38细胞株。
更优选地,所述来源于HCC1143、HCC1599和HCC38细胞株的基因DNA质量比为(1~3)∶(2~5)∶(1~6)。
本发明第二方面提供一种上述标准品的制备方法,所述制备方法包括如下步骤:
(a)获取HCC1143、HCC1599和HCC38细胞株;
(b)分别提取HCC1143、HCC1599和HCC38细胞株中的基因组DNA;
(c)按照配比将不同细胞株的DNA进行混合、浓度测定,得到所述待测混合样品。
(d)对待测样本开展WES检测、HRR-panel检测、ddPCR验证检测,经过数据分析和验证后,得到所述标准品。
本发明第三方面提供一种所述标准品在DNA同源重组修复基因检测试剂盒中的应用。
本发明第四方面提供一种用于DNA同源重组修复基因检测的试剂盒所述试剂盒包括所述标准品。
与现有技术相比,本发明的有益效果至少包括:
1.国内暂时没有同类产品,而市场是有巨大的需求,尤其是在乳腺癌、卵巢癌、前列腺癌的诊疗上;
2.该标准品包含28个相关基因,250个以上突变,突变类型全覆盖(SNV/indel/CNV等),临床注释全覆盖(B/LB/US/LP/P),突变频率全覆盖(0-100%),适合各种检测应用,全方面的检测;
3.采用两种方法学检测验证,NGS侧重大范围的检出,DdPCR侧重检测的准确性;
4.NGS采取高深度(1000x)WES测序,2次检测,完善检出的准确性;
5.同时采取超深度(3500x)的panel捕获子的方法,对HRR相关基因的突变位点精准检测;
6.DdPCR采用高灵敏度的绝对定量,采用两种主流设备平台,对数据进一步精准检测,兼顾不同的研发需求;
7.适用LDT和IVD产品的研发,作为研发过程中的参考品、标准品、质控品,在实际临床样本检测中,作为质控品。
具体实施方式
下面将结合实施例对本发明技术方案的实施例进行详细的描述。以下实施例仅用于更加清楚地说明本发明的技术方案,因此只作为示例,而不能以此来限制本发明的保护范围。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。以下实施例中的定量试验,均设置三次重复实验,数据为三次重复实验的平均值或平均值±标准差。
需要注意的是,除非另有说明,本申请使用的技术术语或者科学术语应当为本发明所属领域技术人员所理解的通常意义。
DNA同源重组修复(Homologous Recombination Repair,HRR)是DNA双链损伤的重要修复方式。HRR是一条涉及到多个步骤的复杂的信号通路,其中关键蛋白为BRCA1和BRCA2。根据ARIEL3的报告,通路主要涉及BRCA1、BRCA2、ATM、ATR、BARD1、BLM、BRIP1、CDK12、CHEK1、CHEK2、FANCA、FANCC、FANCD2、FANCF、FANCI、FANCL、FANCM、MRE11A、NBN、PALB2、PPP2R2A、RAD50、RAD51B、RAD51C、RAD51D、RAD52、RAD54L和RPA1这28个基因。这些基因的很多突变都能引起DNA修复的异常,导致HRD,与肿瘤的发生、诊断、治疗息息相关。
在NCCN的指南中,HRR相关基因的检测已经被写入乳腺癌、卵巢癌、前列腺癌等的指导原则中,市场亟待相关诊断试剂盒的开发。因此围绕HRR相关基因的诊断标准品也是应运而生。
实施例1
1、选择60株肿瘤细胞,具体如表1所示,根据细胞说明培养60种细胞,使用0.25%胰酶(Gibco#25200056)消化细胞后,使用countess II(thermo AMQAX1000)计数,每种细胞收集1×106细胞悬液;
表1
2、采用细胞基因组DNA提取试剂盒(天根生化科技(北京)有限公司)分别提取60种细胞的基因组DNA(具体操作详见厂家的说明书);
3、对提取的gDNA做QC检测:
3.1制备好的DNA使用qubit 3.0定量;
3.1.1将待测样品和QubitTMdsDNA HS Assay kit从冰箱拿出来放至室温;
3.1.2配置染料(Buffer:染料=200:1);
3.1.3在1.5ml离心管中加入18ul QubitTMdsDNA HS Buffer和2ul待测样品,混匀;
3.1.4准备两只QubitTMassay tubes,分别加入190ul染料和10ul标准品1和2,混匀;
3.1.5另取一只QubitTMassay tubes,加入198ul染料和2ul步骤3混匀的样品,混匀;
3.1.6首先用步骤4配置好的标准品对机器进行校准,后检测步骤5的样品;
3.1.7记录检测完的DNA浓度;
3.2琼脂糖凝胶电泳分析DNA降解程度以及是否有RNA污染;
3.3 Nanodrop检测DNA的纯度(OD260/280比值);
3.4样本总量>500ng,电泳无降解,OD260/280比值介于1.8-2.0之间,视为合格样本,可以继续下面的实验;
4、文库构建
样品检测合格后,采用Bioruptor超声打断仪将基因组DNA随机打断至350bp,对打断后的DNA片段进行末端修复、加A尾,并连接上illumina测序通用接头,对其文库进行第一次PCR扩增,随后采用IDT xGen Exome Research Panel v2试剂盒捕获含有目的基因的片段,将捕获的基因片段再进行第二次PCR扩增,得到最终的DNA文库,其中采用KAPA DNA建库试剂盒构建DNA文库;
5、文库质检
文库构建完成后,先使用Qubit3.0进行初步定量,稀释文库至1ng/μl,随后使用Agilent 2100对文库的插入片段长度进行检测,符合预期后,再使用Q-PCR方法对文库的有效浓度进行准确定量(文库有效浓度>2nM),以保证文库质量;
6、上机测序
库检合格后,把不同文库按照有效浓度及目标下机数据量的需求pooling后进行Hiseq测序,测序深度500x WES,测序策略为双末端测序;
7、生物信息分析
下机数据经过处理后获得原始数据,经过过滤去接头、去污染、然后与参考基因组比对;通过比对结果,去除出每个文库中由于PCR扩增引起的重复序列,然后计算出相对于参考基因组的测序深度和覆盖度、单核苷酸位点变异(SNV)、插入/缺失(InDels)等;此外,对于检测出的变异信息还可以进行注释和功能富集等一系列生物信息分析;
8、筛选基因测序数据
针对60株细胞的WES突变数据开展分析,按照如下的原则设计panel,选择其中的原始样本合集:
首先需要一些高频的突变,突变重合度不会很高,因此选择2-3种样本混合为最佳;
28个基因需要全部有突变存在,排除掉40个样本,剩余20个样本待选;
突变类型尽量多样,SNV的同义,错义,剪切变异等等,Indel,CNV需要包括,排除掉10个样本;
临床注释需要包含5类:Benign,Likely Benign,Uncertain Significance,Likely Pathogenic,Pathogenic,其中Pathogenic是越多越好,得到最优化的5个样本;
选择的位点,利于ddPCR开发,主要是考虑重复序列的区段,筛选得到3个样本HCC1143、HCC1599和HCC38细胞株;
9、筛选基因文库比例的确定
将大部分的的突变频率设置为20%,同时兼顾高频和低频,利用拷贝数算法,对3个细胞株的基因文库做质量配对分析,最终选择1~3份HCC1143样本,2~5份HCC1599样本,1~6份HCC38样本的混合样本,根据先前的WES数据,计算理论混样的频率值和拷贝数值;
10、对混合样本的gDNA做QC分析:
10.1制备好的DNA使用qubit 3.0定量;
10.1.1将待测样品和QubitTMdsDNA HS Assay kit从冰箱拿出来放至室温;
10.1.2配置染料(Buffer:染料=200:1);
10.1.3在1.5ml离心管中加入18ul QubitTMdsDNA HS Buffer和2ul待测样品,混匀;
10.1.4准备两只QubitTMassay tubes,分别加入190ul染料和10ul标准品1和2,混匀
10.1.5另取一只QubitTMassay tubes,加入198ul染料和2ul步骤3混匀的样品,混匀;
10.1.6首先用步骤4配置好的标准品对机器进行校准,后检测步骤5的样品;
10.1.7记录检测完的DNA浓度;
10.2琼脂糖凝胶电泳分析DNA降解程度以及是否有RNA污染;
10.3 Nanodrop检测DNA的纯度(OD260/280比值);
10.4样本总量>500ng,电泳无降解,OD260/280比值介于1.8-2.0之间,视为合格样本,可以继续下面的实验。
11、混合样本文库构建
混合样品检测合格后,采用Bioruptor超声打断仪将基因组DNA随机打断至350bp,对打断后的DNA片段进行末端修复、加A尾,并连接上illumina测序通用接头,对其文库进行第一次PCR扩增,随后采用IDT xGen Exome Research Panel v2试剂盒捕获含有目的基因的片段,将捕获的基因片段再进行第二次PCR扩增,得到最终的DNA文库,其中采用KAPA DNA建库试剂盒构建DNA文库;
12、文库质检
文库构建完成后,先使用Qubit3.0进行初步定量,稀释文库至1ng/μl,随后使用Agilent 2100对文库的插入片段长度进行检测,符合预期后,再使用Q-PCR方法对文库的有效浓度进行准确定量(文库有效浓度>2nM),以保证文库质量;
13、上机测序
库检合格后,把文库按照有效浓度及目标下机数据量的需求pooling后进行Hiseq测序,测序深度500x WES,测序策略为双末端测序;
14、生物信息分析
下机数据经过处理后获得原始数据,经过过滤去接头、去污染、然后与参考基因组比对;通过比对结果,去除出每个文库中由于PCR扩增引起的重复序列,然后计算出相对于参考基因组的测序深度和覆盖度、单核苷酸位点变异(SNV)、插入/缺失(InDels)等;此外,对于检测出的变异信息还可以进行注释和功能富集等一系列生物信息分析;
15、委托北京和上海2家企业,开展HRR的pannel超深度检测,2家都已经开发了HRR相关基因的panel,3500x的捕获子,帮助联合定标;
在2次1000x WES和2次3500x panel的数据上,开展重合性分析,同时兼顾第8步中的理论值,确认存在的位点(全部检出),确认突变频率的值(要求多次检测CV在20%以内),得到一份明确的基因和位点清单;
16、对于多次检测存在差异的点,按照如下原则分析:
对于检出与否的差异:首先查看原始的bam,确认是否存在突变点,如果存在,找到被滤掉的原因,如果原因是可以修正的,则修正为检出;如果不存在突变,则记入待定,如果探针未覆盖,则忽略这个检测;
对于检出后频率差异较大(CV大于20%)的位点,首先查看该点的测序深度,如果深度不够,则舍弃这个数值;如果深度足够,则记入待定;
对于修正检出后的频率差异较大,同样先查看该点的测序深度,如果深度不够,则舍弃这个数值;如果深度足够,则记入待定;
17、对16中记入待定的点,按照如下的标准,判断是否开发DdPCR:
如果该位点是这个基因中的唯一位点,则一定要开发;
如果该位点有明确的ACMG的注释,则一定要开发;
如果该位点无任何注释,则选择性开发;
如果该位点无任何注释,而且在内含子的区域,可以不开发;
18、根据第17步的选择清单,设计DdPCR的引物、探针,开展DdPCR检测,考虑到不同的位点,不同的DdPCR检测体系,存在不同的检测限问题,安排在2个DdPCR平台上开展检测,QX200和Micro-Drop100B共同验证清单位点;
19、验证后的结论是:经过1000x WES,3500x Panel和ddPCR共同定标后,得到精准的HRR检测标准品,可以用于HRR检测方法的性能评价。
实施例2
本实施例为一种用于DNA同源重组修复基因检测的标准品,该标准品包括基因panel;
该基因panel包括28种基因,28种基因对应的名称如下:
BRCA1、BRCA2、ATM、ATR、BARD1、BLM、BRIP1、CDK12、CHEK1、CHEK2、FANCA、FANCC、FANCD2、FANCF、FANCI、FANCL、FANCM、MRE11A、NBN、PALB2、PPP2R2A、RAD50、RAD51B、RAD51C、RAD51D、RAD52、RAD54L和RPA1;
上述基因panel来源于HCC1143、HCC1599和HCC38细胞株,且来源于HCC1143、HCC1599和HCC38细胞株的基因DNA的质量比为2∶3∶4。
实施例3
本实施例为实施例2标准品的制备方法,该制备方法包括如下步骤:
(a)获取HCC1143、HCC1599和HCC38细胞株,根据细胞说明培养3种细胞,使用0.25%胰酶(Gibco#25200056)消化细胞后,使用countess II(thermo AMQAX1000)计数,每种细胞收集1×106细胞悬液;
(b)使用细胞基因组DNA提取试剂盒(天根生化科技(北京)有限公司)分别提取HCC1143、HCC1599和HCC38细胞株中的基因组DNA;
(c)按照实施例1中的QC检测方法对基因组DNA进行检测;
(d)检测合格后,采用Bioruptor超声打断仪将基因组DNA随机打断至350bp,对打断后的DNA片段进行末端修复、加A尾,随后,进行第一次PCR扩增,随后采用IDT xGen ExomeResearch Panel v2试剂盒捕获含有目的基因的片段,将捕获的基因片段再进行第二次PCR扩增,得到最终的DNA文库,其中采用KAPA DNA建库试剂盒构建DNA文库;
(d)按照2∶3∶4质量比比将不同细胞株的DNA文库进行混合、浓度测定,得到上述标准品。
本发明中通过在特定的细胞株中分别获得与HRR相关的28种基因,并通过特定比例混合,使得每种基因都包含多样的突变类型以及突变频率全覆盖,进而能够用于HRR检测中的质控。
最后应说明的是:以上各实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述各实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分或者全部技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的范围,其均应涵盖在本发明的权利要求和说明书的范围当中。
Claims (6)
1.一种用于DNA同源重组修复基因检测的标准品,其特征在于,包括基因panel;
所述基因panel包括28种基因,所述28种基因对应的名称如下:
BRCA1、BRCA2、ATM、ATR、BARD1、BLM、BRIP1、CDK12、CHEK1、CHEK2、FANCA、FANCC、FANCD2、FANCF、FANCI、FANCL、FANCM、MRE11A、NBN、PALB2、PPP2R2A、RAD50、RAD51B、RAD51C、RAD51D、RAD52、RAD54L和RPA1。
2.根据权利要求1所述的标准品,其特征在于,所述基因panel来源于肿瘤细胞株,所述肿瘤细胞株包括HCC1143、HCC1599和HCC38细胞株。
3.根据权利要求2所述的标准品,其特征在于,所述来源于HCC1143、HCC1599和HCC38细胞株的基因DNA质量比为(1~3)∶(2~5)∶(1~6)。
4.权利要求1~3任一所述的标准品的制备方法,其特征在于,包括如下步骤:
(a)获取HCC1143、HCC1599和HCC38细胞株;
(b)分别提取HCC1143、HCC1599和HCC38细胞株中的基因组DNA;
(c)按照配比将不同细胞株的DNA进行混合、浓度测定,得到所述待测混合样品;
(d)对待测样本开展WES检测、HRR-panel检测、ddPCR验证检测,经过数据分析和验证后,得到所述标准品。
5.权利要求1~3任一所述的标准品在DNA同源重组修复基因检测试剂盒中的应用。
6.一种用于DNA同源重组修复基因检测的试剂盒,其特征在于,所述试剂盒包括权利要求1~3任一所述的标准品。
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