CN112522380A - 一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法 - Google Patents
一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法 Download PDFInfo
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Abstract
本发明公开了一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法,其步骤包括,(1)样本收集;(2)panel设计;(3)文库构建;(4)上机测序;(5)数据分析注释。本专利发明基于目标区域捕获高通量测序技术流程,通过选取与MF、LDS、SGS和ACC相关的特异有效基因集合使用多重PCR捕获流程,一次性进行高通量捕获测序分析。克服了现有技术需要进行八次核苷酸检测,不仅费时,还耗费患者的费用。本发明与现有技术相比具有针对性强、成本低、流程快等优势。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种基于高通量测序技术的马凡及其类综合征基因突变的检测方法。
背景技术
马凡综合征(Marfan syndrome,MFS)是一种主要表现为骨骼、眼和心血管系统受累的症状,易引发主动脉夹层和/或主动脉破裂。MFS患病率0.065‰~0.2‰。此外,还有一些表型与MFS类似但患病率更低的其他综合征,如Loeys-Dietz综合征(LDS)、Shprintzen-Goldberg综合征(SGS)和比尔斯综合征(ACC)。MF、LDS、SGS和ACC都是单基因遗传性结缔组织疾病,临床表型类似且有较大的异质性。MFS主要为常染色体显性遗传,编码原纤维蛋白1的FBN1基因为其主要致病基因。目前报道的FBN1基因相关突变超过1800个,符合临床诊断标准的MFS患者检出FBN1基因突变的比例为70%~93%。LDS目前缺乏公认的临床诊断标准,检测出致病基因突变是确诊和分型最有力的依据;致病基因有TGFBR1、TGFBR2、SMAD3、TGFB2和TGFB3。约90%的Shprintzen-Goldberg综合征患者可检出SKI基因突变。ACC又称先天性挛缩蜘蛛指(趾)患者的致病基因是FBN2。2019年我国首版《罕见病诊疗指南》和《单基因遗传性心血管疾病基因诊断指南》都提及马凡及其类综合征的鉴别诊断依赖于致病基因的鉴别,确定致病位点是个体化治疗和预防疾病遗传的基础。当前临床常用的基因检测方法有多种,如Sanger测序、基因分型、高通量测序、Array CGH。Sanger测序作为广泛应用的技术,具有快速、准确、简便等优点,但存在通量低、相对成本高、自动化程度低等问题,难以满足一次性检测多基因多变异的要求。基因分型有多种技术,大多具备快速、准确、低成本的优点,十分适合大规模检测已知变异,但只能检测到SNP等单核苷酸的已知变异。ArrayCGH是检测拷贝数变异(CNV)的金标准,但不能同时检测SNP、Indel变异。
MF、LDS、SGS和ACC临床表型高度异质且有不同程度的重合,仅靠临床症状往往难以区分确诊。此外,疾病发病过程隐匿,早期诊断困难。MFS主要为常染色体显性遗传,编码原纤维蛋白1的FBN1基因为其主要致病基因。目前报道的FBN1基因相关突变超过1800个,符合临床诊断标准的MFS患者检出FBN1基因突变的比例为70%~93%。Loeys-Dietz综合征的致病基因有TGFBR1、TGFBR2、SMAD3、TGFB2和TGFB3,SGS的致病基因是SKI,ACC的致病基因是FBN2。传统技术很难一次性排查这些基因上的变异,新一代测序技术提供了新的解决方案。随着测序技术的发展,高通量测序技术的成本、周期和性能都有了极大的改进,全外显子组、全基因组测序已经成了科研发现的常规方法,可同时检测基因组上所有类型的变异,但针对性不强,且数据量大,各项成本比较高。临床应用通常追求快速、经济、简便,选取一组有效基因集合进行高通量测序以降低成本,成为一种可用于检测遗传性主动脉疾病突变基因的有效方法。
发明内容
本发明要解决的技术问题是当前缺乏有效、低成本检测MF、LDS、SGS和ACC的基因突变方法,针对该技术问题采用多重PCR捕获技术,将选取的特定有效基因集合利用扩增原理予以捕获,然后通过高通量测序平台进行检测,利用生物信息分析流程和ACMG遗传解读指南,检测解读MF、LDS、SGS和ACC的基因突变,以辅助临床对测遗传性主动脉疾病进行精确确诊以及治疗。本方法获得的结果可应用于MF、LDS、SGS和ACC的鉴别诊断、疾病的预防和干预、个体化治疗和产前诊断。
为实现上述目的,本发明采用的技术方案是:
一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法,其步骤包括,
(1)样本收集:
选取马凡及其类综合征相关的8个致病基因FBN1、TGFBR1、TGFBR2、SMAD3、TGFB2、TGFB3、SKI、FBN2;
(2)panel设计:
试剂盒由7管试剂构成,
其中5管试剂保存在-20℃,包括1管IGT-I7 Index(10uM)试剂、1管IGT-I5 Index(10uM)试剂、1管Primer pool试剂、1管IGT-EM808 polymerase mixture试剂和1管Enhancer buffer NB(1N)试剂;
另2管试剂保存在4℃,包括1管Enhancer buffer M试剂和1管YF buffer B试剂;
设计的试剂盒中,Primer pool试剂共包含213个扩增子,扩增子的序列为1-213,每个扩增子均包括一个正向引物和一个反向引物,正向引物和反向引物的序列为SEQ IDNo.1-SEQ ID No.426;
(3)文库构建:
(3.1)第1轮多重PCR反应
在PCR管中按下表配方配制反应液,用枪轻柔地上下吹吸混匀;
运行PCR仪,放入上述PCR管,按下列程序进行反应:先95℃210s,之后执行循环程序:98℃10s、60℃5min循环18次,最后72℃延伸5min;
(3.2)磁珠纯化合并产物
(3.2.1)向30ul步骤(3.1)处理的PCR产物中加入27ul室温平衡后的AMPure XP磁珠,用移液器吸打混匀20次;
(3.2.2)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.2.3)彻底移除上清,将PCR管从磁力架取下,向管内加入50ul YF buffer B并用移液器吸打混匀20次;
(3.2.4)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.2.5)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分比浓度80%乙醇溶液,静置30s;
(3.2.6)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分比浓度80%乙醇溶液,静置30s后彻底移除上清;
(3.2.7)室温静置3min,使残留乙醇彻底挥发;
(3.2.8)将PCR管从磁力架取下,加入24μl的Nuclease-free water,移液器轻轻吸打重悬磁珠,避免产生气泡,之后室温静置2min;
(3.2.9)将PCR管重新置于磁力架上,静置3min;
(3.2.10)用移液器吸取13.5μl上清液,转移到新的200μl的PCR管内,管内移入的上清液即为多重PCR产物;
(3.3)第2轮接头序列PCR反应
在PCR管中按下表配方配制反应液,用枪轻柔地上下吹吸混匀,其中,PCR productmixture为步骤(3.2.10)获得的纯化后的多重PCR产物;
运行PCR仪,放入上述PCR管,按下列程序进行反应:先95℃210s,之后执行循环程序:98℃20s、68℃1min、72℃30s循环9次,最后72℃延伸5min;
(3.4)第2轮磁珠纯化
(3.4.1)取30ul步骤(3.3)处理的PCR反应体系,并加27ul室温平衡后的AMPure XP磁珠,用移液器吸打混匀20次;
(3.4.2)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.4.3)彻底移除上清,将PCR管从磁力架取下,向管内加入50ul YF buffer B并用移液器吸打混匀20次;
(3.4.4)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.4.5)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分数为80%乙醇溶液,静置30s;
(3.4.6)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分数为80%乙醇溶液,静置30s后彻底移除上清;
(3.4.7)室温静置3min,使残留乙醇彻底挥发;
(3.4.8)将PCR管从磁力架取下,加入24μl的Nuclease-free water或者1×TEbuffer(pH 8.0),用移液器轻轻吸打混匀20次,重悬磁珠,避免产生气泡,室温静置2min;
(3.4.9)将PCR管重新置于磁力架上,静置3min;
(3.4.10)用移液器吸取20μl上清液,转移到新的200μl的PCR管内,管内移入的上清液即为制备好的多重PCR文库;
(3.5)文库定量
取2ul步骤(3.4.10)获得的多重PCR文库,并使用3.0Fluorometer(QubitdsDNA HS Assay Kit)进行文库浓度测定,记录文库浓度;唾液gDNA或血液gDNA所构正常文库的浓度范围为5-40ng/ul;
(4)上机测序
将步骤(3.5)获得的多重PCR文库使用Hiseq Xten PE150进行测序获得基因序列表,上机数据量为150Mb;
(5)数据分析注释:测序数据下机后,对下机数据进行生物信息学分析,检测基因上的变异;
(5.1)下机数据Fastq格式质量控制:对每个样本文库和lane产生的每对原始Fastq序列raw reads,使用软件cutAdapt去除测序接头和包含大量‘N’碱基的序列reads,得到满足质控的序列Clean reads,使用软件fastqc统序列计Clean reads基本信息,包括序列和碱基数量、GC含量和分布、测序错误率和分布、碱基质量分布;
(5.2)比对Alignment:使用软件bwa,把序列Clean reads与人类参考基因组GRCh38比对,得到序列Clean reads在参考基因组上的比对信息文件BAM;
(5.3)比对信息文件BAM预处理:使用picard和GATK软件把同一样本不同文库、lane产生的比对信息文件BAM文件合并一起,按照基因组坐标排序,验证比对信息文件BAM文件,去除PCR过程中产生的重复序列,校正碱基质量值,得到预处理后的比对文件CleanBAM;使用picard软件统计比对文件Clean BAM,得到质控信息;所述质控信息包括对目标区域捕获芯片的覆盖度、深度、捕获效率、均一性,可比对序列和碱基的数量,插入片段的长度分布;
(5.4)变异检测:使用GATK软件检测基因上的小片段变异,包括单核苷酸多态性(SNP)和插入、缺失(Indel);对检测变异进行过滤,SNP过滤条件为:QD<3.37||FS>31.397||SOR>10.419||MQ<20.0||MQRankSum<-12.49||ReadPosRankSum<-3.721,Indel过滤条件为:QD<5.2||FS>52.254||SOR>9.044||ReadPosRankSum<-5.504;
对满足过滤条件的变异进行注释,标注出对应的基因、转录本、变异类型、功能、在正常人群中的频率;
统计变异结果,得到变异数量、长度分布、各类型变异数量、人群变异频率信息。
为了获得更好的技术效果,在步骤(3.1)之前先将待检测gDNA的浓度稀释到同一浓度,之后转移到PCR8联管中。
为了获得更好的技术效果,在步骤(3.5)后还有步骤(3.6)文库质量检测,
取1ul步骤(3.4)获得的多重PCR文库,并使用Qsep100全自动核酸蛋白分析系统进行文库片段长度和纯度测量,正常文库的靶片段分布区间在300bp-420bp之间;
将步骤(3.4)获得的多重PCR文库使用Aligent 2100及QPCR进行文库检测;
步骤(3.4)获得的多重PCR文库Aligent 2100片段结果在250bp-350bp之间即为合格,qpcr结果大于10nM即为合格;
将检测合格的多重PCR文库用于步骤(4)进行测序获得基因序列表。
为了获得更好的技术效果,还包括步骤(6)结果解读:解释变异SNP和变异Indel与疾病之间的关系;
依据ACMG/AMP指南通过使用软件REO-HIT把变异分成5类:致病、疑似致病、意义未明、疑似良性、良性。
为了获得更好的技术效果,还包括步骤(7)出具报告,通过Sanger法测序逐个验证上面检测出致病、疑似致病、意义未明、疑似良性的测序结果,并出具报告。
本专利发明基于目标区域捕获高通量测序技术流程,通过选取与MF、LDS、SGS和ACC相关的特异有效基因集合使用多重PCR捕获流程及下述实施方案进行高通量捕获测序分析,旨在通过基因检测技术解读MF、LDS、SGS和ACC的致病基因的变异情况。多基因变异分析的结果参考美国遗传学会(ACMG)、分子病理学会(AMP)和中华医学会心血管病学分会精准心血管病学学组等发布的指南解读变异。该发明与现有技术相比具有针对性强、成本低、流程快等优势。
试剂生产厂家:Illumina公司。
附图说明
图1为本发明实施例遗传变异分析解读流程;
图2为本发明实施例Qsep100全自动核酸蛋白分析系统质检文库结果;
图3为本发明实施例检测技术流程。
具体实施方式
下面结合附图和具体实施方式,进一步详细阐述本发明的内容。
一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法,其步骤包括,
(1)样本收集:
选取马凡及其类综合征相关的8个致病基因FBN1、TGFBR1、TGFBR2、SMAD3、TGFB2、TGFB3、SKI、FBN2;
(2)panel设计:
试剂盒由7管试剂构成,
其中5管试剂保存在-20℃,包括1管IGT-I7 Index(10uM)试剂、1管IGT-I5 Index(10uM)试剂、1管Primer pool试剂、1管IGT-EM808 polymerase mixture试剂和1管Enhancer buffer NB(1N)试剂;
另2管试剂保存在4℃,包括1管Enhancer buffer M试剂和1管YF buffer B试剂;
根据步骤(1)筛选出的目的基因总计8个,进行panel设计,设计区域为各基因外显子区,并允许外显子区两侧各延伸15bp;
设计的试剂盒中,Primer pool试剂共包含213个扩增子,扩增子的序列为1-213,每个扩增子均包括一个正向引物和一个反向引物,正向引物和反向引物的序列为SEQ IDNo.1-SEQ ID No.426,详细引物序列见附录3;
IGT-I7 Index的位置信息请见附录1《96个IGT-I7 Index位置信息》;
IGT-I5 Index的信息请见附录2;
试剂生产厂家:Illumina公司;
(3)文库构建:
(3.0)将所有待检测gDNA的浓度稀释到同一浓度,并转移到PCR8联管中,一方面是便于排枪操作,另一方面是为了加入相同起始量的gDNA,降低最终文库的浓度差异,便于混合文库;请在PCR管壁上方或者管盖上进行反应编号标记,防止高温或其他原因导致记号消失,避免后续产物混合操作失误,造成样本交叉污染;
(3.1)第1轮多重PCR反应
在PCR管(或排管、或PCR板)里按下表配方配制反应液,用枪轻柔地上下吹吸混匀;
运行PCR仪,放入上述PCR管,按下列程序进行反应:先95℃210s,之后执行循环程序:98℃10s、60℃5min循环18次,最后72℃延伸5min;
(3.2)磁珠纯化合并产物
(3.2.1)向30ul步骤(3.1)处理的PCR产物中加入27ul室温平衡后的AMPure XP磁珠,用移液器吸打混匀20次;
(3.2.2)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.2.3)彻底移除上清,将PCR管从磁力架取下,向管内加入50ul YF buffer B并用移液器吸打混匀20次;
(3.2.4)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.2.5)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分比浓度80%乙醇溶液,静置30s;
(3.2.6)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分比浓度80%乙醇溶液,静置30s后彻底移除上清,建议使用10μl移液器移除底部残留乙醇溶液;
(3.2.7)室温静置3min,使残留乙醇彻底挥发;
(3.2.8)将PCR管从磁力架取下,加入24μl的Nuclease-free water,移液器轻轻吸打重悬磁珠,避免产生气泡,之后室温静置2min;
(3.2.9)将PCR管重新置于磁力架上,静置3min;
(3.2.10)用移液器吸取13.5μl上清液,转移到新的200μl的PCR管内,管内移入的上清液即为多重PCR产物;
(3.3)第2轮接头序列PCR反应
在PCR管(或排管、或PCR板)内按下表配方配制反应液,用枪轻柔地上下吹吸混匀,其中,PCR product mixture为步骤(3.2.10)获得的纯化后的多重PCR产物;
运行PCR仪,放入上述PCR管,按下列程序进行反应:先95℃210s,之后执行循环程序:98℃20s、68℃1min、72℃30s循环9次,最后72℃延伸5min;
(3.4)第2轮磁珠纯化
(3.4.1)取30ul步骤(3.3)处理的PCR反应体系,并加27ul室温平衡后的AMPure XP磁珠,用移液器吸打混匀20次;
(3.4.2)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.4.3)彻底移除上清,将PCR管从磁力架取下,向管内加入50ul YF buffer B并用移液器吸打混匀20次;
(3.4.4)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.4.5)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分数为80%乙醇溶液,静置30s;
(3.4.6)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分数为80%乙醇溶液,静置30s后彻底移除上清,建议使用10μl移液器移除底部残留乙醇溶液;
(3.4.7)室温静置3min,使残留乙醇彻底挥发;
(3.4.8)将PCR管从磁力架取下,加入24μl的Nuclease-free water或者1×TEbuffer(pH 8.0),用移液器轻轻吸打混匀20次,重悬磁珠,避免产生气泡,室温静置2min;
(3.4.9)将PCR管重新置于磁力架上,静置3min;
(3.4.10)用移液器吸取20μl上清液,转移到新的200μl的PCR管内,管内移入的上清液即为制备好的多重PCR文库;
(3.5)文库定量
取2ul步骤(3.4.10)获得的多重PCR文库,并使用3.0Fluorometer(QubitdsDNA HS Assay Kit)进行文库浓度测定,记录文库浓度;唾液gDNA或血液gDNA所构正常文库的浓度范围为5-40ng/ul;
(3.6)文库质量检测
取1ul步骤(3.4)获得的多重PCR文库,并使用Qsep100全自动核酸蛋白分析系统进行文库片段长度和纯度测量,正常文库的靶片段分布区间在300bp-420bp之间;检测结果见图2;
将步骤(3.4)获得的多重PCR文库使用Aligent 2100及QPCR进行文库检测;
步骤(3.4)获得的多重PCR文库Aligent 2100片段结果在250bp-350bp之间即为合格,qpcr结果大于10nM即为合格;
(4)上机测序
将步骤(3.6)获得的合格的多重PCR文库使用Hiseq Xten PE150进行测序获得基因序列表,上机数据量为150Mb;
(5)数据分析注释:测序数据下机后,对下机数据进行生物信息学分析,检测基因上的变异;
(5.1)下机数据Fastq格式质量控制:对每个样本文库和lane产生的每对原始Fastq序列raw reads,使用软件cutAdapt去除测序接头和包含大量‘N’碱基的序列reads,得到满足质控的序列Clean reads,使用软件fastqc统序列计Clean reads基本信息,包括序列和碱基数量、GC含量和分布、测序错误率和分布、碱基质量分布;
(5.2)比对Alignment:使用软件bwa,把序列Clean reads与人类参考基因组GRCh38比对,得到序列Clean reads在参考基因组上的比对信息文件BAM;
(5.3)比对信息文件BAM预处理:包括使用picard、GATK软件把同一样本不同文库、lane产生的比对信息文件BAM文件合并一起,按照基因组坐标排序,验证比对信息文件BAM文件,去除PCR过程中产生的重复序列,校正碱基质量值,得到预处理后的比对文件CleanBAM;使用picard软件统计比对文件Clean BAM,得到质控信息;所述质控信息包括对目标区域捕获芯片的覆盖度、深度、捕获效率、均一性,可比对序列和碱基的数量,插入片段的长度分布;
(5.4)变异检测:使用GATK软件检测基因上的小片段变异,包括单核苷酸多态性SNP和插入、缺失Indel;
对检测变异进行过滤,SNP过滤条件为:QD<3.37||FS>31.397||SOR>10.419||MQ<20.0||MQRankSum<-12.49||ReadPosRankSum<-3.721,Indel过滤条件为:QD<5.2||FS>52.254||SOR>9.044||ReadPosRankSum<-5.504;
对满足过滤条件的变异进行注释,获得变异信息,所述变异信息包括基因、转录本、变异类型、功能、在正常人群中的频率;
统计变异结果,得到变异数量、长度分布、各类型变异数量、人群中出现的比例;
(6)结果解读:解释变异SNP和变异Indel与疾病之间的关系;
依据ACMG/AMP指南(Genet Med.2015 May;17(5):405–424.)通过使用软件REO-HIT把变异分成5类:致病、疑似致病、意义未明、疑似良性、良性;
软件REO-HIT处理过程如图1所示:
具体步骤为,
加载数据库:加载ClinVar、CGD和OMIM数据库;
收集变异信息:对步骤(5.4)获得的变异信息逐个读取,获得基本变异信息、人群变异频率信息、疾病变异信息、变异功能预测信息以及变异保守性;
处理解读证据:
i)通过数据库对照收集的变异信息,依次判断每个变异是否满足ACMG/AMP指南(Genet Med.2015 May;17(5):405–424.)所述致病性或者良性证据;
其中,致病性证据包括:极强致病PVS、强致病PS、中等致病PM、弱致病PP;
良性证据包括:独立良性BA、强良性BS、弱良性BP;
ii)组合变异证据,根据ACMG/AMP组合规则给每个变异分类为:致病、疑似致病、意义未明、疑似良性、良性;
(7)出具报告
通过Sanger法测序逐个验证上面检测出致病、疑似致病、意义未明、疑似良性的测序结果,并出具报告。
对比例
Sanger测序是检测DNA突变的金标准,但由于其灵敏度有限,且无法对多个目标同时进行并行探测。
若采用Sanger测序检测FBN1、TGFBR1、TGFBR2、SMAD3、TGFB2、TGFB3、SKI和FBN2基因需要每个外显子分别进行Sanger测序,成本昂贵。
本发明所用目标区域捕获高通量测序涉及到FBN1、TGFBR1、TGFBR2、SMAD3、TGFB2、TGFB3、SKI和FBN2基因外显子的靶向富集,用Illumina XTen的平行测序,只需一次即可实现检测上述全部基因外显子。
本专利发明的特点为:一种基于高通量测序技术的MF、LDS、SGS和ACC的基因突变检测方法和应用,特征为针对MF、LDS、SGS和ACC进行基因检测,同时在实验过程中使用标签化技术提高结果检测的准确度。
基因列表如表1所述,目标区域为各基因外显子和两侧各延伸15bp的区间。
检测方法:
(1)将待检测基因组DNA打断,主带为300-400bp
(2)将打断的DNA片段进行纯化、末端修复、加接头并进行PCR扩增
(3)捕获建库过程中使用标签化接头,降低测序噪音污染,矫正PCR错误,提高结果的准确性,尤其是针对低频的突变更易被检测;
(4)将扩增产物与目标区域捕获芯片进行杂交,扩增、纯化后得到目标DNA测序文库
(5)使用测序文库进行测序,得到目前基因上的序列
(6)使用测序序列进行生物信息学分析,包括序列质量控制、序列比对、比对文件预处理以及检测SNP、Indel变异、变异过滤和注释
(7)对变异结果进行解读,并分为致病、疑似致病、意义未明、疑似良性、良性5类。
检测过程中要求测序数据量达到150Mb以上,测序深度达到150X,覆盖度高于99.9%.
本发明涉及到一种基于高通量测序技术的MF、LDS、SGS和ACC的基因突变检测方法和应用,采用多重PCR技术,可以同时捕获第三部分所示的8个基因的全部编码区域及侧翼区域。准确解读变异与疾病关系,为MF、LDS、SGS和ACC的筛查、早期诊断、鉴别诊断、生育指导、运动建议提供重要依据。具有全面、成本低等优势。
附录1:
IGT-I7 Index的位置信息,使用BOX1,96孔板,
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
A | A01 | A02 | A03 | A04 | A05 | A06 | A07 | A08 | A09 | A10 | A11 | A12 |
B | B01 | B02 | B03 | B04 | B05 | B06 | B07 | B08 | B09 | B10 | B11 | B12 |
C | C01 | C02 | C03 | C04 | C05 | C06 | C07 | C08 | C09 | C10 | C11 | C12 |
D | D01 | D02 | D03 | D04 | D05 | D06 | D07 | D08 | D09 | D10 | D11 | D12 |
E | E01 | E02 | E03 | E04 | E05 | E06 | E07 | E08 | E09 | E10 | E11 | E12 |
F | F01 | F02 | F03 | F04 | F05 | F06 | F07 | F08 | F09 | F10 | F11 | F12 |
G | G01 | G02 | G03 | G04 | G05 | G06 | G07 | G08 | G09 | G10 | G11 | G12 |
H | H01 | H02 | H03 | H04 | H05 | H06 | H07 | H08 | H09 | H10 | H11 | H12 |
。
附录2:IGT-I5 Index序列信息
IGT-I5 Index端的分样序列与测序平台相关:
1.MiniSeq,NextSeq,HiSeq 3000/4000和Hiseq X Ten测序平台,请用Index列的分样序列;
2.MiSeq,HiSeq 2000/2500和NovaSeq测序平台,请用Inprimer列的分样序列;
3.如果是单端测序,仅以IGT-I7 Index列分样序列拆分数据即可。
Name | Indcx分样序列 | Inprimcr分样序列 |
IGT-15-31# | GTAGAGGA | TCCTCTAC |
IGT-15-32# | CCGCCTTA | TAAGGCGG |
IGT-15-33# | ATAGTACG | CGTACTAT |
IGT-I5-34# | TTCTGCCT | AGGCAGAA |
。
附录3:
Primer pool试剂共包含213个扩增子,扩增子的序列为1-213,每个扩增子均包括一个正向引物和一个反向引物,正向引物和反向引物的序列为SEQ ID No.1-SEQ IDNo.426,具体见下表,
SEQUENCE LISTING
<110> 心脑血管防治教育部重点实验室
赣南医学院第一附属医院
段, 艳宇
刘, 子由
<120> 一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法
<130> 2020
<160> 426
<170> PATENTIN VERSION 3.5
<210> 1
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 1
GTTTTTCTTT TAATTATTTG GTCTCTGGAT GG 32
<210> 2
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 2
AAGGGATCAG CTACCTCCAC TT 22
<210> 3
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 3
AGCAAAGATG GCTGTCTTCT CA 22
<210> 4
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 4
TGTGTATGCA GCATAAGGCA GA 22
<210> 5
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 5
CTCCAACCAT GACCAGGAAG AG 22
<210> 6
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 6
AACGAATGCC TCAGCGCTC 19
<210> 7
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 7
AGCCATGCAT CTTGAGAGTG AG 22
<210> 8
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 8
TAGGATGTGT AGGGGCCAGA TT 22
<210> 9
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 9
TTCTTATCCC AACAGCAGAG GAA 23
<210> 10
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 10
GTTGGCTTGA CTCAAATGCC TC 22
<210> 11
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 11
CCACTTGAGG ATAAGCCATC AGA 23
<210> 12
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 12
ACACTGAAGT GACCCCCTAC ATA 23
<210> 13
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 13
CACAGCAGCA TTCCGATTTG G 21
<210> 14
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 14
GCTTTCCCCT CTTGCTTCTT CT 22
<210> 15
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 15
GTCACTTCTG ATGCACTCAA AGC 23
<210> 16
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 16
AGTCAGGTAA TTAAGGCAGA TATATGCA 28
<210> 17
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 17
TGTTCCCAGG ATCAGTACAC GTA 23
<210> 18
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 18
CATCATGTTT TGGACACATT CCTGG 25
<210> 19
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 19
CTGAGAATCC AGCACAGGCA A 21
<210> 20
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 20
TTCCTCTGGT TTCTGGGCTT G 21
<210> 21
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 21
GCATTGAAAG CCCAAAGCCT TC 22
<210> 22
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 22
ACTCAGTTGC CCTTTGTGTG TC 22
<210> 23
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 23
GCATGATTCC TTGAGTGGTC TCT 23
<210> 24
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 24
TGCTGGGATT ATGACATCTT TGGA 24
<210> 25
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 25
AGAATCAAAT GAAGCTTTCA ACAGCA 26
<210> 26
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 26
TGTCTTCTAA GTTCTCACTT AAGATGCT 28
<210> 27
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 27
CGCCAAGTGT GTATCAAGTA GC 22
<210> 28
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 28
GATCATGTGC TGTCCTGTCA CT 22
<210> 29
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 29
CCCAACAATT CATGGGTAAT TTTTCAAC 28
<210> 30
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 30
TCACCAACCC TCCAATCCTT TTT 23
<210> 31
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 31
ACCTTATCAT CCTACCAGGA CCAT 24
<210> 32
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 32
CACACAACTT GAATTTCCTT GGGT 24
<210> 33
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 33
AGAAAGGAGA ACTGGCTGGA GT 22
<210> 34
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 34
TCTGCCTGAT GCTTTTGTGT TT 22
<210> 35
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 35
TGATTTTGAT GCCAGTGGAG GT 22
<210> 36
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 36
CTCTGCATTT TCTTAGTATT TACATTAGTT GC 32
<210> 37
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 37
GAAATGTGGA ATGCCTGGCT TC 22
<210> 38
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 38
TTGGTTTTAA ATACCACCCT TTCTGTT 27
<210> 39
<211> 34
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 39
ACATGTATCA ATCTATAATT ATGATACCAA TCTC 34
<210> 40
<211> 34
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 40
GGAAAGTACT CAATGATATC AAATAGCTAC ATAT 34
<210> 41
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 41
AAAGCCTGGG CCCTAAACTA C 21
<210> 42
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 42
GAGTATTGGA GGGGACAGAC ATC 23
<210> 43
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 43
TTCCCTATGA GGTTCACGCA AC 22
<210> 44
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 44
CCATAACACA GAGGGAAGTT ACCG 24
<210> 45
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 45
GCCATCAAAG CTTCATGGAA TCC 23
<210> 46
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 46
AAGGCTGTCC TGAGACTCAT TTG 23
<210> 47
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 47
GGCAATTGGC CATGGAAAAC G 21
<210> 48
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 48
ATCCGCCTGG AAACCTGCTT 20
<210> 49
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 49
CCCTATCGGA CATGCTGAAT TTTG 24
<210> 50
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 50
AGATATAGAT GAATGTGAAG TGTTCCC 27
<210> 51
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 51
GACAGCTTTA TCCAGTCCGA GT 22
<210> 52
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 52
TGTCAGAACT GCAAAGTCTG GA 22
<210> 53
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 53
TTCTTTTGCA GGAAAAGCTG ACA 23
<210> 54
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 54
TAGGCCCAAG ACTAGATTTT AGCAG 25
<210> 55
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 55
CAGGCAATGT TTCAGAAAAT GGGTA 25
<210> 56
<211> 34
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 56
CCTAAGGTCA TTACATTTAT TGTAGTGTTA TATT 34
<210> 57
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 57
AAGACCTCAA TGGTGGCAGA AG 22
<210> 58
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 58
GTGACAGTGT GATGACAGAT GC 22
<210> 59
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 59
CATAAGGAGG AGAAAAGGCA CGT 23
<210> 60
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 60
CCTTCGTAAG CTTACTCTTC TGGTC 25
<210> 61
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 61
TATGTCCCAC ATTCCACGTC AG 22
<210> 62
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 62
GGAACCCAGA AAGTCTTAGA ATTATGAG 28
<210> 63
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 63
ACTTGAACAA TGCAAGAAAA ATAACTAGAT G 31
<210> 64
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 64
CTGTTGTGTT TTGTTTTGTT GTGTTTT 27
<210> 65
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 65
GGAACTGACT TACACAAACC ATGC 24
<210> 66
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 66
CTGAGTCCTT CTACTGACGA ATGG 24
<210> 67
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 67
GAGAATGGCT CTCCAGAGCA A 21
<210> 68
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 68
ACAGGACAGG CCCATGTTTT 20
<210> 69
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 69
TTCAGGAAGT AGCCATGCAG AC 22
<210> 70
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 70
AAGCGTCTCA GCTCTCTCCT TA 22
<210> 71
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 71
TGCAGGAAAG AGGAAAGCCA A 21
<210> 72
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 72
CATGTGCTAA CAGACCTCTG GT 22
<210> 73
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 73
CTTGAAACTT GGGAGACCCA CA 22
<210> 74
<211> 16
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 74
CAAGAGGCGG CGGGAG 16
<210> 75
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 75
AATAGGTTCC AGCCACTGGC TT 22
<210> 76
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 76
CAGAAGCCAA TGTGAGTCTT GC 22
<210> 77
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 77
AGACATCAGG AGAAACTAAC TTCTGAC 27
<210> 78
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 78
CCTTGTCTTC CCATTCTAAT GAAAAACA 28
<210> 79
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 79
CGCAGAGCCA CATTCATTGA TG 22
<210> 80
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 80
AGCAAGTGGC CAGATCCAAT G 21
<210> 81
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 81
ACAAGGATTC ACCAGCTGGA TC 22
<210> 82
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 82
GGAATTTTAA CCCCTCTTTG CCC 23
<210> 83
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 83
GTGGAGTTCT TACAGGCAAA GGA 23
<210> 84
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 84
GATGCACAGT CACGCTGTAT TTC 23
<210> 85
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 85
AGATTCCCTG CAAGTATTTT TGGA 24
<210> 86
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 86
CAGACAATCG GGAAGGGTAC T 21
<210> 87
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 87
TTAGGAAAGT GCGGTGCCAA 20
<210> 88
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 88
AAGGAAGGAG CTCCATCCTC TA 22
<210> 89
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 89
AAGGGAAGCT TTGAGGGACA TC 22
<210> 90
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 90
GGTAGGTTCC CTTTTGTTGC TG 22
<210> 91
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 91
TGGTACCTAT ATTCATGGCT ATACAGTG 28
<210> 92
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 92
CTTCACGTTT AAAAAATACC TTGTTATTCA CT 32
<210> 93
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 93
CTTACATCAT GGCCAGTCTG CA 22
<210> 94
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 94
AGGAAAGCAA CTGAAGGGTG TC 22
<210> 95
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 95
TTCTGCTAAG TCCAGTGGAC AC 22
<210> 96
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 96
CATGCCAGTG GGAACCTCTT 20
<210> 97
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 97
ACTGACTTCC TTTGCTGATG CA 22
<210> 98
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 98
TGCTCTTTAG CCACTGTAAC CG 22
<210> 99
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 99
AGACAAACTC TTGGGTAGGC ATG 23
<210> 100
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 100
TGAGAGGCTT TGTTGACTGG AC 22
<210> 101
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 101
GAAAGTTCTG ACAATGCCGT CATG 24
<210> 102
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 102
CCAATTATTG TTCTTTGCTG ACCCC 25
<210> 103
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 103
GAACTTGTGA GCTCTCTTCC TCT 23
<210> 104
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 104
TCAGGCCATT CCAAAATGTG AAG 23
<210> 105
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 105
CCCAAGGAAA TTCAAGTTGT GTGT 24
<210> 106
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 106
CAGACGGGCA GAGTAACAAC TAA 23
<210> 107
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 107
ACACAGTATG CTTGCTTCTC TGA 23
<210> 108
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 108
TTGGGCCCTG TTCTTTTATG GT 22
<210> 109
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 109
CCTAATCTCA TCAAGCCCAG CA 22
<210> 110
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 110
AACCGAGGAA GAGTAACGTG TG 22
<210> 111
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 111
GCAGTCCTTG ATAAGCAACC TCT 23
<210> 112
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 112
CCAAAAGACA TTTGTGCTGA GCC 23
<210> 113
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 113
CAGTGCTTAT GACTAACAAG ACAAGATG 28
<210> 114
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 114
GACTGCGGTC AGTTAATGTT TTCTC 25
<210> 115
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 115
GACTGCGGTC AGTTAATGTT TTCTC 25
<210> 116
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 116
GACAGACATC AATGAATGTG AGCTG 25
<210> 117
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 117
GACCACAAGT AAATGGTGTG AAAGTC 26
<210> 118
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 118
GTCAAGATGG ACACCCAGCA AT 22
<210> 119
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 119
TTTAAAGGAC GTCCCCTCTC CT 22
<210> 120
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 120
ACCTCCTGAC TGCTTGCTCA TA 22
<210> 121
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 121
CACTCCTCGC ATTCCTCAGT AC 22
<210> 122
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 122
GGAAGCCGTG TGGCTCTATT TA 22
<210> 123
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 123
CACTTGAATG ACCCCCTAGT GT 22
<210> 124
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 124
GTTCTGGTTG CTATTCAGGC AC 22
<210> 125
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 125
CATTGGAGTG GTATAGGAAC CACA 24
<210> 126
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 126
TGCTATTTTT GTCTATAATT CCAAGGTGT 29
<210> 127
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 127
GGCATTCCAA AAGATAGCAA AGTACAC 27
<210> 128
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 128
GCAAAGTAGA TACAGGCAAA GTTTGG 26
<210> 129
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 129
CATCCCAGAT ACATGGCACA GT 22
<210> 130
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 130
AAGGGCAGGA TCTACCTGTT CT 22
<210> 131
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 131
CCTCTCTCAT AAGGTTAGCC ATGATG 26
<210> 132
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 132
TCCTATCTTC CCCATTTTCA AGGG 24
<210> 133
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 133
ACTGCAATGG AAGGAGAGGA CT 22
<210> 134
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 134
TGAGTTTGCA AATGGAGGGA GG 22
<210> 135
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 135
ACAGAATTAC AACAGACCCT TGGT 24
<210> 136
<211> 30
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 136
TGTCAGATTA AGTACTGATG AAAGATACCA 30
<210> 137
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 137
GCTGGGATGG GATATTCTGC A 21
<210> 138
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 138
TCAGCGATGT GTGTGTGTGT 20
<210> 139
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 139
ACTACACCCC CCAACTGCAA 20
<210> 140
<211> 30
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 140
TGGTAACATA ATTGTGGACA AATTATCACA 30
<210> 141
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 141
CCGTGCGGAT ATTTGGAATG AAG 23
<210> 142
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 142
TTCCTCTGCA TGATGGTTCC TG 22
<210> 143
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 143
CCCAGGTAAT CGAAGAAAAT CCATC 25
<210> 144
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 144
CTGTGAGCTG TTGCAATCTA TGC 23
<210> 145
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 145
CCCCATGCAA CCAACACAAC 20
<210> 146
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 146
GTCTGCCAGG ATTCATCTTG CT 22
<210> 147
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 147
CGAGGTTTGC TGGGGTGAG 19
<210> 148
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 148
GAGAAAGAGC AGGAGCGAGC 20
<210> 149
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 149
TCGAGATAGG CCGTTTGTAT GTG 23
<210> 150
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 150
TGGACAAGTC ACTTCTTGCC TC 22
<210> 151
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 151
TGATGGTCTA TATCTGCCAC AACC 24
<210> 152
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 152
TCTCACATTC TAGCAAGTTG GCTTA 25
<210> 153
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 153
CTCTAGAGAA GAACGTTCGT GGTT 24
<210> 154
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 154
ATGGGTCTAA TCTACATGAG AGACATC 27
<210> 155
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 155
TGATTGGTAT TACCTTTTAA GCAGTCATG 29
<210> 156
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 156
AAGGGAAAAA AGGTGATTTC AGAAGATATT AA 32
<210> 157
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 157
GATCTTTTAA TGCCTTGGCA TTAGCT 26
<210> 158
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 158
TGCTTACTAA GCAGAAGCAG TTTAGA 26
<210> 159
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 159
AACCTTGAGA TTTTTTCTAA GAATCTTTCT CT 32
<210> 160
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 160
TGGTCCTGAT TGCAGCAATA TGT 23
<210> 161
<211> 30
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 161
GTTTTTGTCG TTGTTGATGT TTATTTCACT 30
<210> 162
<211> 34
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 162
GTTGTCATAT CATAAATTAA GTCTTTCAAC GTAG 34
<210> 163
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 163
GAAACATGTA ATATTGTTGA TTGTGTTGAG T 31
<210> 164
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 164
AGGATGTTTT CATGACGTAA CATTACAG 28
<210> 165
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 165
GCCCAACCGA AATGTTAATT CTGT 24
<210> 166
<211> 33
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 166
TGAAAATTTA AAGCTTAAAT AATAGAACTG CTT 33
<210> 167
<211> 33
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 167
TGAAAATTTA AAGCTTAAAT AATAGAACTG CTT 33
<210> 168
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 168
CTCATGACAA ACTACTGGGG GA 22
<210> 169
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 169
TCTTATCCAG ACCAATGGAA AATGGT 26
<210> 170
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 170
AGCAGATCTG AAGAAAAAAG GAGAGT 26
<210> 171
<211> 34
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 171
TGGCAGTTGG ATAATCATTT AATATATCTT TCTC 34
<210> 172
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 172
TGTAAAAAGG GGAAAAGAAA GAATAACTTC T 31
<210> 173
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 173
ACCTACCACA TCCAACTCCT TC 22
<210> 174
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 174
CTTCCAGGAT GATGGCACAG T 21
<210> 175
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 175
GCTGAGGTCT ATAAGGCCAA GC 22
<210> 176
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 176
GTACTCCTGT AGGTTGCCCT TG 22
<210> 177
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 177
TCACCTCCAC AGTGATCACA CT 22
<210> 178
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 178
GGTAAAGGGG ATCTAGCACT AGCT 24
<210> 179
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 179
CTGCACATGC CATTCTCAGT GA 22
<210> 180
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 180
TCTGCCACCT AAGAGGCAAC TT 22
<210> 181
<211> 16
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 181
GCCAGGGGTC CGGGAA 16
<210> 182
<211> 17
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 182
CTGTCAAGCG CAGCGGA 17
<210> 183
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 183
TCAAAACAGT TTCACTTTCC TGTCATC 27
<210> 184
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 184
AGTGAGGGAG CATGACTAAA AATAGAA 27
<210> 185
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 185
CCCTCGCTTC CAATGAATCT CT 22
<210> 186
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 186
AGGTCCCACA CCCTTAAGAG AA 22
<210> 187
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 187
GAAGCTGAGT TCAACCTGGG AA 22
<210> 188
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 188
GATCTTGACT GCCACTGTCT CA 22
<210> 189
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 189
GGGAAACAAT ACTGGCTGAT CAC 23
<210> 190
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 190
AGGTTAGGTC GTTCTTCACG AG 22
<210> 191
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 191
AAATGATGGG CCTCACTGTC TG 22
<210> 192
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 192
CACTACACAA TGATGCTGGT CCA 23
<210> 193
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 193
GGTGCCCTTT GGATCTCTTT CC 22
<210> 194
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 194
TGAGAGGGGC AGCCTCTTT 19
<210> 195
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 195
GGAGGAGAAA TGGTGCGAGA AG 22
<210> 196
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 196
TCTCTCCCTC TTCCCATCTC CA 22
<210> 197
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 197
CCTCTCTTTC TGCCCCTCCC 20
<210> 198
<211> 18
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 198
TGCTGTCCCC ACAGGCAG 18
<210> 199
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 199
CATGGTGTGC ATGTGTGATG TC 22
<210> 200
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 200
AGGAAGGGAT GGAAGGGATG AA 22
<210> 201
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 201
AGTAGCCCAC CCTGTGTCC 19
<210> 202
<211> 17
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 202
GCGGGGAATG GAGCCAC 17
<210> 203
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 203
GGTCCAGGAC TTGCTTTATC CA 22
<210> 204
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 204
CTGATGTAGG CAGCACCCAT AA 22
<210> 205
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 205
TCTAGGAAGG GCTGTATTGT CCT 23
<210> 206
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 206
GAGAAACTCT GACAGATCTC TGGC 24
<210> 207
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 207
ACTGAGCCAC CTCTGCTCT 19
<210> 208
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 208
AGAGATTGGG GCCACAGGA 19
<210> 209
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 209
TCTTTCTGCT GTGTTGGGCT AC 22
<210> 210
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 210
TTCCAGTTGT GTGCACAAGG AG 22
<210> 211
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 211
CCTGTCCAGT CTAACCTGAA TCTC 24
<210> 212
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 212
TGAGAAAGGT GGACTCTCTC CA 22
<210> 213
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 213
ACTTGTGTAA CCCCCTGGAG A 21
<210> 214
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 214
TCCAATTTTC TCCACCTCCT GC 22
<210> 215
<211> 15
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 215
CCGTCGAGCC CAGCC 15
<210> 216
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 216
GGATGGTGAT GCACTTGGTG 20
<210> 217
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 217
ATGGACCAGT TCATGCGCAA 20
<210> 218
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 218
GAAGAAGGGC GGCATGTCTA TT 22
<210> 219
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 219
ATAGTTCTGT GCCAGGCATC TC 22
<210> 220
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 220
CAAGTTCCCC ACAGGAGACA AA 22
<210> 221
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 221
GAATGCCAAC TCAGCCTTTT CTC 23
<210> 222
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 222
CATCTTGGGA GGAAAAGAGA GAGTG 25
<210> 223
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 223
GTGAAGCTAA ATGTTTATTA CCCAAATGC 29
<210> 224
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 224
TGCAATTACT TGGTTTTACT TTTCTTTCC 29
<210> 225
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 225
TGTTGTCTCT CCTCTCCTGT GT 22
<210> 226
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 226
CATCGTTGTC GTCGTCATCA TC 22
<210> 227
<211> 30
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 227
CAAACAACTC TCCTTGATCT ATACTTTGAG 30
<210> 228
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 228
ACTGGTGAGC TTCAGCTTGC 20
<210> 229
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 229
AAGAGTACTA CGCCAAGGAG GT 22
<210> 230
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 230
AAGGACAGGA GAACGGGAAA AG 22
<210> 231
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 231
AGTCTTCTCT GAAAGCACAA TGGA 24
<210> 232
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 232
CAACAACAAC AACAACAACA ACAAATG 27
<210> 233
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 233
CCTCCCAAGA TGTTCAGTAT CCCTA 25
<210> 234
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 234
TTCCCTCCCC CACCTCATAT G 21
<210> 235
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 235
GTGAGGGTGG TGAATCAGCT T 21
<210> 236
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 236
ATGGTAACCC AAGAACAGAG GC 22
<210> 237
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 237
CCAGTGCCTC AGATGGCAT 19
<210> 238
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 238
GAGAGCATTC ATGAAGTTTC TTTTATTGG 29
<210> 239
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 239
TCTTGAGGTC TGGTAAGGGT CT 22
<210> 240
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 240
CCATCACACA GAGGTGCTTT TC 22
<210> 241
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 241
TGGTTTCTGC TCTGAGAGAG GA 22
<210> 242
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 242
AGCACACTGT TCCTGCATGT 20
<210> 243
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 243
TGATAGGGGA CGTGGGTCAT C 21
<210> 244
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 244
TCTCTTCCTC TCCAGGCCTT G 21
<210> 245
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 245
AGGCAGTGGT GGTTCTCTCT 20
<210> 246
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 246
CCAACCTCTC ACTGACATGT CC 22
<210> 247
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 247
AGTAGGCAGG CAGTAGATGT TG 22
<210> 248
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 248
AGGAGATTGT CACTTTCCTT CCC 23
<210> 249
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 249
AGGGACCTGA GGTTCATTCT GA 22
<210> 250
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 250
CTGTGCTCTG CATTGTGACA TC 22
<210> 251
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 251
ACACCCCAGC GAGAATTTGG 20
<210> 252
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 252
TGGAAGCCAT TAGGGGACAG A 21
<210> 253
<211> 18
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 253
CCGGGCCCTT CTTCATGC 18
<210> 254
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 254
GAAGGGCAGG ATGCCCATG 19
<210> 255
<211> 17
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 255
GCTGGGCCTG GAGCTCA 17
<210> 256
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 256
GTGTAATCCT GGCTCAGCAG G 21
<210> 257
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 257
CCACAGGTGC CAACAAAACC 20
<210> 258
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 258
CAAGGAGAAG GGCCCAGTAC 20
<210> 259
<211> 18
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 259
AGAGGCACCT TCCCGACA 18
<210> 260
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 260
TCCTTGTCCT CCTCTGGGG 19
<210> 261
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 261
GGCACTTCCA TGACTTTGTT TCTG 24
<210> 262
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 262
TCTCTTTGGC TTCCTTGGTG TC 22
<210> 263
<211> 17
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 263
TGGGCAGTGA CCCCGAG 17
<210> 264
<211> 17
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 264
CGAGTACTTG GCGCGCA 17
<210> 265
<211> 16
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 265
GCGCCGCTGA CCACTC 16
<210> 266
<211> 16
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 266
GTTGTCGGCG GCGCTG 16
<210> 267
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 267
GTTCCACCTG AGCTCCATGA G 21
<210> 268
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 268
TCTCGCCTTC CAGTACGGT 19
<210> 269
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 269
ACGAGCTCCA CATCTACTGC T 21
<210> 270
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 270
CCTTACACTT GCCGAAGCAC T 21
<210> 271
<211> 18
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 271
AGAACCGGAC CTGCCACT 18
<210> 272
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 272
GAGACCAGAG CCTGTAGTCC A 21
<210> 273
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 273
TTCTCCTGGT CACTCACACA GA 22
<210> 274
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 274
TCAGAAACAC CTGTGAATGG CG 22
<210> 275
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 275
CCAGCAGAAG GTTGTGAGCA 20
<210> 276
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 276
ACAGGGAGGA GGCACAGAAA 20
<210> 277
<211> 16
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 277
AGGCGGAGCT GGAGCA 16
<210> 278
<211> 18
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 278
CATGGGGCAG CCAGGAAG 18
<210> 279
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 279
ATGAAAGAGG CCAACGAGTC AC 22
<210> 280
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 280
GCTTCTGCTC CAAGGCCTTT 20
<210> 281
<211> 17
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 281
GTGATCGGCC GTGAGCC 17
<210> 282
<211> 16
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 282
CGCGCCGAGA AAGCGG 16
<210> 283
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 283
GGGATGCTAG TGATTTCCAG TGT 23
<210> 284
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 284
TCAGCCTAGA GAGTGTCGAC AT 22
<210> 285
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 285
CCGTAGGGAT AAAATTTTGT GGCAA 25
<210> 286
<211> 30
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 286
GTCAGTGATA AAGTCTAACA ATACTTTGCT 30
<210> 287
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 287
GTGTTAGAGC AGCCGTAATT GC 22
<210> 288
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 288
GCTTCCCCGT GTAAGAGTTT CT 22
<210> 289
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 289
TTTTTCAGAT TATACCAATT TGTCTTGGAA G 31
<210> 290
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 290
GGAAAGTTTG ACGCTCTGCT TC 22
<210> 291
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 291
AGGGTCACGA TTGTCAGAGA CT 22
<210> 292
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 292
TGGAAGGAGG AAAGAGTCTG GT 22
<210> 293
<211> 33
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 293
TGTGTTGATT ATATGAATAA ATTTCCTCAA CTC 33
<210> 294
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 294
TTGCCATTTC AGACAATCGA CAG 23
<210> 295
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 295
CTTCTGTGAT GACATCATGA ACAGATG 27
<210> 296
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 296
AGCTTGTAAA CTATAATTTG ACAGTTTTCT CA 32
<210> 297
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 297
AGTTCGAACT ACTCCCGAGT CT 22
<210> 298
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 298
TGAGCACATT CCTGTCCATT CC 22
<210> 299
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 299
GGGTACCTGA GAGACCTTTT ACTG 24
<210> 300
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 300
TGAGGAAATT CACATGCAGT TCT 23
<210> 301
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 301
CTGGTACACA AAGTGCTGTC TGA 23
<210> 302
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 302
GAGAAATGTT CTGTCCAAAC AGTGTTAC 28
<210> 303
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 303
GTGGGCCTCA GAATAAATGT TACTC 25
<210> 304
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 304
CAGAGGACCC GAGTTGAATT CAT 23
<210> 305
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 305
AGAAGCAATA AAGGACTGAA TGAAGTAC 28
<210> 306
<211> 33
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 306
CTGAATGAGG AAAATCTAAG TAATTCCATA AGT 33
<210> 307
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 307
GTGCTCAGTG CCAAAGAATG AG 22
<210> 308
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 308
GTTGATCATC AAAGTGTGAC GTGA 24
<210> 309
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 309
GGATCCCAGA TTATTATATT TTGTGGTACT C 31
<210> 310
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 310
GGAATTTGAG AAATTTAAGA GTGCATTGC 29
<210> 311
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 311
CAACTGTGCT CTAATTAATC TCAGAGTTC 29
<210> 312
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 312
AGAGAATCAC CTATGCTTTG TTCAGT 26
<210> 313
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 313
TCTGGGTGAC AATGGGATTA GC 22
<210> 314
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 314
CTTGCTCATT GTCTCAGCCA CT 22
<210> 315
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 315
TGTAGCAAAC ACTCATCACG GTT 23
<210> 316
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 316
GGTAACCTGG GCATTTTAAA TGCT 24
<210> 317
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 317
GAAAATGAGT TGGAATCCTG GTGAC 25
<210> 318
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 318
AGAGCAAGGA GAAAATGTGT CATG 24
<210> 319
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 319
GCCTTAGCAA AGGATATTTA CATTTGC 27
<210> 320
<211> 34
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 320
TGCATCTTTA CTTTAAAATG TTACCTATAC AAAA 34
<210> 321
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 321
GTGCATGTGT GTGTATAAAT GTTTATCC 28
<210> 322
<211> 27
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 322
CCAAGTGGAA CCAGATAGAA AACAAAA 27
<210> 323
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 323
CATATGAGTT TCAGGCCTGC TTG 23
<210> 324
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 324
CTCTGGTGAA GCACCTTTCT GA 22
<210> 325
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 325
CGCACGAATG AGTCTGTGCT A 21
<210> 326
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 326
GCTTGGAAGG CCAAGTCTAT CTC 23
<210> 327
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 327
AAGAAGGACC AAGGCGCTG 19
<210> 328
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 328
TTCGCATGGA GCAGTGTTAC T 21
<210> 329
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 329
GGTTTTGGAC TAGCCACTGC T 21
<210> 330
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 330
CATCTTTGCT CCTCTGCTTG C 21
<210> 331
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 331
AGTGGCAGGA GAAGTTTCCA AG 22
<210> 332
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 332
AGGTAAGAGC TCCCCAACCT TT 22
<210> 333
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 333
GCACAAATAA GGCGATGAAG ACAG 24
<210> 334
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 334
TGCTGCCCAC TGTAATAGAT GCT 23
<210> 335
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 335
CGATTATGTC ACGTGATTAT TATAAAGTTG AG 32
<210> 336
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 336
GGTAGTTTTG GAGTTATGAA ATGACTTGTA TA 32
<210> 337
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 337
ATTTTGCACA GTGGGGCCAT 20
<210> 338
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 338
TACCCAGTAA CTGCTTGGCT TC 22
<210> 339
<211> 34
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 339
AAAAATGTAT ATCCTTTTAA AATCTTTTGC AAGG 34
<210> 340
<211> 34
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 340
TCCATTTCTT ATCTTACTGT GTTTATCTAT AAGT 34
<210> 341
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 341
CATTCCAATT TGATCCTGTA ATCTAATAGC T 31
<210> 342
<211> 33
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 342
TGCATGTTGT AATGATTAAA TATAATTGTG CTT 33
<210> 343
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 343
AACGCCAGGA GAAAAGCCAT 20
<210> 344
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 344
TCACCTGTGC TTAATGCCAA CT 22
<210> 345
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 345
CATACCTGTT TGGGCTGTTT TGTC 24
<210> 346
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 346
AAGGCCTTTC TCAATCATCT TCTCC 25
<210> 347
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 347
ATGGTGTGCA CAGGCAGACA 20
<210> 348
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 348
ACCTGTCTTT CTTTCTTCTC CCACA 25
<210> 349
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 349
TCAAAAGATC ACAGGATTTA TGAGACCT 28
<210> 350
<211> 33
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 350
ACCTCTCACA TATACTATAA TTTTGATGAC TGA 33
<210> 351
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 351
CGCAACTATG CGTCCAAATG G 21
<210> 352
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 352
CTACCTATCT GTTACGTGAC CGC 23
<210> 353
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 353
GATACTCGGG CGCTAGAAAC C 21
<210> 354
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 354
GTCTTTGATC ACTCCCTCTC CG 22
<210> 355
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 355
ACTGGCTGTG CTAGGATTTG AG 22
<210> 356
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 356
AAGGAATGGG CTCAGCTACT TG 22
<210> 357
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 357
CTTAGTTCCA GGATGTGCTC CT 22
<210> 358
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 358
CTAGTCCTCT GCTTGTGCTG TA 22
<210> 359
<211> 20
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 359
TATGTGCTGA GGCTGAAGGC 20
<210> 360
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 360
TGAGAATGAA TGCTCCAATC CCA 23
<210> 361
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 361
GTGAGAAAAG CAAGTGCGAT GG 22
<210> 362
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 362
GTGACATAAA GGTTTTGCCG GG 22
<210> 363
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 363
CTGAACATAA ATACCAGGAC ATTCTTCTG 29
<210> 364
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 364
AGTCTACTGT GATTTTCACT GGCTT 25
<210> 365
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 365
TGAGTGACTT GCAGGAAACA GT 22
<210> 366
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 366
TACCCTTGCA TATATGTTAA CTCCTGAT 28
<210> 367
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 367
ACGAGATTGC GACTACTGGA TG 22
<210> 368
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 368
AGGAGGAAAT TCTTGACTGC CC 22
<210> 369
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 369
GAGTGGAGGC ATTACATAAG CAGA 24
<210> 370
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 370
CAGAAGACCA CACCTGTGAC TTAA 24
<210> 371
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 371
CTCACCAGCA GAATATCAGA AGCA 24
<210> 372
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 372
CATCTTTGTG GCAGTTTCTT ATTCTTCTT 29
<210> 373
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 373
ACTATATGTT ACATAGTAAA GTTAGCATGA GT 32
<210> 374
<211> 32
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 374
CATGTATATT GTTCATCCTT TCTTAAGTTC TT 32
<210> 375
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 375
AGCGTGAACT GTGACAGTGA AG 22
<210> 376
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 376
AGCTGGCTAG TGGACAGTGA TA 22
<210> 377
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 377
AAAACATAAC ACCACAAAGA CTGCTT 26
<210> 378
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 378
GTAGCGTCTT GTGAAACAAT ATACATGT 28
<210> 379
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 379
TTCACTCCCT GGAGCCACA 19
<210> 380
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 380
AGCAGATTAG AGAAACACAA CTGTGA 26
<210> 381
<211> 33
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 381
CAGAAACCAT CTAAATGCTA TATATGTATA CCA 33
<210> 382
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 382
CAGAACAACA TATATTTGAC CAAATCTGTT C 31
<210> 383
<211> 19
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 383
GCAGTCAGCA GATGCACGA 19
<210> 384
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 384
CTACAGCTGT GTGGGCTTCA T 21
<210> 385
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 385
CAGCACTGAG CATTTTAGGC ATC 23
<210> 386
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 386
AGGTGTTTGT TTTAAGTGCA CTGTC 25
<210> 387
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 387
CCAGTTGGTT TTCTTCCTCT TCTTC 25
<210> 388
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 388
CAACTGTAAG CTCCTTTGTT GCTC 24
<210> 389
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 389
GAACACAACT TAGCTGGTAA CTGAC 25
<210> 390
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 390
TCTGAGGGGG CAGAAAAATG C 21
<210> 391
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 391
TGTTCTATGA CCATCCCGTC AGA 23
<210> 392
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 392
GACAAACGAT AATCTTTCCA GGTGC 25
<210> 393
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 393
GATGTTGAAA GTTGGCCTTT GACA 24
<210> 394
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 394
ACTGTGATGA GCACATACTA AGATATTGA 29
<210> 395
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 395
TGTTTTCTAT CTGGTTCCAC TTGGT 25
<210> 396
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 396
GACAATGTGG TTGCAATGCT GT 22
<210> 397
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 397
CCTGGTCTTT ACCAGTTGTG CT 22
<210> 398
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 398
TCTCCCACGC TGTATGTGTG AA 22
<210> 399
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 399
AGGGCAGTCC TTTCAAACAG AG 22
<210> 400
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 400
AACCGGCTCT ACATTCTGGC T 21
<210> 401
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 401
CCATGCGGAA CTTTCCAGGA A 21
<210> 402
<211> 21
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 402
ATGGAGTCCA TTCCAGGAGG T 21
<210> 403
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 403
GCTCAAAAGA TGTATAGTAT CTTCCAAGG 29
<210> 404
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 404
CTTCTTCTTG AGCATACCAT GGTTG 25
<210> 405
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 405
GTAAGGCCTG AGATCTGTGC TT 22
<210> 406
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 406
TCCAGAAACA CATGGCAGTT CT 22
<210> 407
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 407
CATGGGATTA AACTATATGA AATGTTCTTG C 31
<210> 408
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 408
GTCTTTACTA GACCTGCATT TATCAACAA 29
<210> 409
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 409
CAACTGTGAA AATGGCACAT GTGA 24
<210> 410
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 410
CTCTATCACT GCCTCAAGTC ATTGT 25
<210> 411
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 411
GGGGAAGCTG AAGTAATTCT TCCA 24
<210> 412
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 412
AAGGCCAGTG TTCAAAGTCA CT 22
<210> 413
<211> 29
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 413
GGAACATTTT CATATGTGAA AGCAAACTG 29
<210> 414
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 414
CTGACTGGCC TTCTCTCAAA CTTTA 25
<210> 415
<211> 22
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 415
AGTCCACAGG AAAACTGACC AC 22
<210> 416
<211> 24
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 416
GGAATATCGC AGACTTTGCA TGGA 24
<210> 417
<211> 23
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 417
GTCTGTGCTG AGGAGACTAA CAG 23
<210> 418
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 418
CAGTGATTGT TGTAAGAACC TGAGG 25
<210> 419
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 419
CAATTAGGCA TGCTTCCCAA AGTAG 25
<210> 420
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 420
CTGAAAACCA GTGTCATCTG TGTTC 25
<210> 421
<211> 31
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 421
GACTTCTTCA GAAAGTAAAG TCTCATTTCT C 31
<210> 422
<211> 26
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 422
TGTGCTTGGA GACTGGCTAT ATAATG 26
<210> 423
<211> 25
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 423
TGCTCCAAAG CTTAAGAAAA ATGCA 25
<210> 424
<211> 28
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 424
ACAGCTTTCT TGTTTTGTAA ACTATGGT 28
<210> 425
<211> 17
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 425
CCCAAGCCGG AGCCCTA 17
<210> 426
<211> 18
<212> DNA
<213> 人工序列(ARTIFICIAL SEQUENCE)
<400> 426
TCCTGTGGCT GGGCTGTG 18
Claims (5)
1.一种基于高通量测序技术检测马凡及其类综合征相关突变基因的方法,其步骤包括,
(1)样本收集:
选取马凡及其类综合征相关的8个致病基因FBN1、TGFBR1、TGFBR2、SMAD3、TGFB2、TGFB3、SKI、FBN2;
(2)panel设计:
试剂盒由7管试剂构成,
其中5管试剂保存在-20℃,包括1管IGT-I7 Index(10uM)试剂、1管IGT-I5 Index(10uM)试剂、1管Primer pool试剂、1管IGT-EM808 polymerase mixture试剂和1管Enhancer buffer NB(1N)试剂;
另2管试剂保存在4℃,包括1管Enhancer buffer M试剂和1管YF buffer B试剂;
设计的试剂盒中,Primer pool试剂共包含213个扩增子,扩增子的序列为1-213,每个扩增子均包括一个正向引物和一个反向引物,正向引物和反向引物的序列为SEQ ID No.1-SEQ ID No.426;
(3)文库构建:
(3.1)第1轮多重PCR反应
在PCR管、排管或PCR板中按下表配方配制反应液,用枪轻柔地上下吹吸混匀;
运行PCR仪,放入上述PCR管,按下列程序进行反应:先95℃210s,之后执行循环程序:98℃10s、60℃5min循环18次,最后72℃延伸5min;
(3.2)磁珠纯化合并产物
(3.2.1)向30ul步骤(3.1)处理的PCR产物中加入27ul室温平衡后的AMPure XP磁珠,用移液器吸打混匀20次;
(3.2.2)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.2.3)彻底移除上清,将PCR管从磁力架取下,向管内加入50ul YF buffer B并用移液器吸打混匀20次;
(3.2.4)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.2.5)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分比浓度80%乙醇溶液,静置30s;
(3.2.6)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分比浓度80%乙醇溶液,静置30s后彻底移除上清;
(3.2.7)室温静置3min,使残留乙醇彻底挥发;
(3.2.8)将PCR管从磁力架取下,加入24μl的Nuclease-free water,移液器轻轻吸打重悬磁珠,避免产生气泡,之后室温静置2min;
(3.2.9)将PCR管重新置于磁力架上,静置3min;
(3.2.10)用移液器吸取13.5μl上清液,转移到新的200μl的PCR管内,管内移入的上清液即为多重PCR产物;
(3.3)第2轮接头序列PCR反应
在PCR管、排管或PCR板内按下表配方配制反应液,用枪轻柔地上下吹吸混匀,其中,PCRproduct mixture为步骤(3.2.10)获得的纯化后的多重PCR产物;
运行PCR仪,放入上述PCR管、排管或PCR板,按下列程序进行反应:先95℃210s,之后执行循环程序:98℃20s、68℃1min、72℃30s循环9次,最后72℃延伸5min;
(3.4)第2轮磁珠纯化
(3.4.1)取30ul步骤(3.3)处理的PCR反应体系,并加27ul室温平衡后的AMPure XP磁珠,用移液器吸打混匀20次;
(3.4.2)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.4.3)彻底移除上清,将PCR管从磁力架取下,向管内加入50ul YF buffer B并用移液器吸打混匀20次;
(3.4.4)室温孵育5min后,将PCR管置于DynaMag-96Side磁力架上3min;
(3.4.5)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分数为80%乙醇溶液,静置30s;
(3.4.6)移除上清,PCR管继续放置在磁力架上,向管内加入180ul体积百分数为80%乙醇溶液,静置30s后彻底移除上清;
(3.4.7)室温静置3min,使残留乙醇彻底挥发;
(3.4.8)将PCR管从磁力架取下,加入24μl Nuclease-free water或者1×TE buffer(pH8.0),用移液器轻轻吸打混匀20次,重悬磁珠,避免产生气泡,室温静置2min;
(3.4.9)将PCR管重新置于磁力架上,静置3min;
(3.4.10)用移液器吸取20μl上清液,转移到新的200μl的PCR管内,管内移入的上清液即为制备好的多重PCR文库;
(3.5)文库定量
取2ul步骤(3.4.10)获得的多重PCR文库,并使用3.0 Fluorometer(QubitdsDNA HS Assay Kit)进行文库浓度测定,记录文库浓度;唾液gDNA或血液gDNA所构正常文库的浓度范围为5-40ng/ul;
(4)上机测序
将步骤(3.5)获得的多重PCR文库使用Hiseq Xten PE150进行测序获得基因序列表,上机数据量为150Mb;
(5)数据分析注释:测序数据下机后,对下机数据进行生物信息学分析,检测基因上的变异;
(5.1)下机数据Fastq格式质量控制:对每个样本文库和lane产生的每对原始Fastq序列raw reads,使用软件cutAdapt去除测序接头和包含大量‘N’碱基的序列reads,得到满足质控的序列Clean reads,使用软件fastqc统序列计Clean reads基本信息,包括序列和碱基数量、GC含量和分布、测序错误率和分布、碱基质量分布;
(5.2)比对Alignment:使用软件bwa,把序列Clean reads与人类参考基因组GRCh38比对,得到序列Clean reads在参考基因组上的比对信息文件BAM;
(5.3)比对信息文件BAM预处理:使用picard和GATK软件把同一样本不同文库、lane产生的比对信息文件BAM文件合并一起,按照基因组坐标排序,验证比对信息文件BAM文件,去除PCR过程中产生的重复序列,校正碱基质量值,得到预处理后的比对文件Clean BAM;使用picard软件统计比对文件Clean BAM,得到质控信息;所述质控信息包括对目标区域捕获芯片的覆盖度、深度、捕获效率、均一性,可比对序列和碱基的数量,插入片段的长度分布;
(5.4)变异检测:使用GATK软件检测基因上的小片段变异,包括单核苷酸多态性(SNP)和插入、缺失(Indel);对检测变异进行过滤,SNP过滤条件为:QD<3.37||FS>31.397||SOR>10.419||MQ<20.0||MQRankSum<-12.49||ReadPosRankSum<-3.721,Indel过滤条件为:QD<5.2||FS>52.254||SOR>9.044||ReadPosRankSum<-5.504;
对满足过滤条件的变异进行注释,标注出对应的基因、转录本、变异类型、功能、在正常人群中的频率;
统计变异结果,得到变异数量、长度分布、各类型变异数量、人群中出现的比例。
2.如权利要求1所述检测马凡及其类综合征相关突变基因的方法,其特征在于,在步骤(3.1)之前先将待检测gDNA的浓度稀释到同一浓度,之后转移到PCR8联管中。
3.如权利要求1或2所述检测马凡及其类综合征相关突变基因的方法,其特征在于,在步骤(3.5)后还有步骤(3.6)文库质量检测,
取1ul步骤(3.4)获得的多重PCR文库,并使用Qsep100全自动核酸蛋白分析系统进行文库片段长度和纯度测量,正常文库的靶片段分布区间在300bp-420bp之间;
将步骤(3.4)获得的多重PCR文库使用Aligent 2100及QPCR进行文库检测;
步骤(3.4)获得的多重PCR文库Aligent 2100片段结果在250bp-350bp之间即为合格,qpcr结果大于10nM即为合格;
将检测合格的多重PCR文库用于步骤(4)进行测序获得基因序列表。
4.如权利要求1或2所述检测马凡及其类综合征相关突变基因的方法,其特征在于,还包括步骤(6)结果解读:解释变异SNP和变异Indel与疾病之间的关系;
通过软件REO-HIT依据ACMG/AMP指南通过使用软件REO-HIT把变异分成5类:致病、疑似致病、意义未明、疑似良性、良性。
5.如权利要求4所述检测马凡及其类综合征相关突变基因的方法,其特征在于,还包括步骤(7)出具报告,通过Sanger法测序逐个验证上面检测出致病、疑似致病、意义未明、疑似良性的测序结果,并出具报告。
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