全外显子及靶向文库捕获测序在多囊肾病基因诊断中的应用比较

目的比较全外显子和靶向文库捕获高通量测序对多囊肾病相关基因的检测效率。方法对6份多囊肾病标本(包括一份低比例嵌合变异标本)分别进行全外显子捕获或靶目标捕获2种方法建立文库,Illumina Hi Seq 2000仪器连续双向测序。结果以PKD1、PKD2和PKHD1 3个基因作为目标序列,靶向捕获法平均测序深度为190倍,5倍以上测序深度占全部有效序列的85.59%,靶区域覆盖度95%以上,但PKD1第一外显子仍有200~300 bp区域不能覆盖;全外显子捕获法平均测序深度为28.34倍,5倍以上测序深度占全部有效序列的55.35%,目标区域覆盖度较低,PKD1外显子覆盖度小于40%。结论靶向文库捕获测序法具有较髙的敏感性、准确性,更适合PKD基因变异的检测,但高通量测序技术对PKD1基因检测仍有不足之处。     

三种人全外显子组捕获探针的性能比较

目的比较不同平台3个全外显子组探针的性能,为以后建立标准流程提供数据支撑。 方法提取人EDTA全血或FFPE组织gDNA,通过酶切方法将gDNA片段化,并为每一个样本的片段化后的DNA加上特异序列的接头,利用IDT、Human Exome和MedExomed三种不同的液相探针的方法将目标基因捕获出来,用Illumina的NextSeq 500二代测序仪进行测序;分析测序数据,比较不同捕获探针的靶向覆盖率、捕获特异性、变异检出能力等。 结果本研究所选入组的3种探针,IDT和MedExome展示了对CCDS和Refseq数据库很好的覆盖率(均>95%);都表现了对靶区域很高的覆盖率(均>99%);IDT探针对血样gDNA捕获特异性(76.96%±0.75%,n=6),明显高于Human Exome (67.63%±1.62%,n=6) (P<0.001);IDT对FFPE标准品捕获特异性为84.48%±0.64%(n=3),明显高于MedExome的71.07%±0.91%(n=3)(P<0.01)。变异输出数量上,Human Exome最高。 结论IDT探针的捕获特异性明显高于其它2种,而在变异输出数量上,Human Exome探针最高。     

无精子症患者基因检测分析

目的:利用全外显子测序技术,筛选无精子症患者相关基因,丰富男性不育基因库。方法:抽取20例无精子症患者外周血,提取DNA,使用杂交捕获方法构建DNA文库,采用高通量测序技术检测人类全外显子组中20099个基因的外显子区及旁侧内含子区(20bp),将测序数据与人类基因组hg19参考序列进行比对,筛选变异基因,变异位点进行Sanger测序验证。结果:共计筛选出26个基因43个变异位点,排除15个常染色体隐性遗传的单个变异位点及13个与精子运动相关的变异位点,剩余11个基因的15个变异位点可能与精子发生障碍相关,其中包括FAM71B、STARD9、CLTCL1、PCBP3、S100PBP等5个在睾丸组织高表达基因,SYCE3、EFCAB6、DDX4、KDM5D、RGS22、MTL5等6个基因可能与精子发生障碍相关。结论:通过研究筛选到可能影响男性不育的基因,为男性不育的基因诊断研究提供参考。     

93例耳聋患儿耳聋基因检测结果

目的分析93例耳聋患儿耳聋基因携带情况,为预防遗传性耳聋提供依据。方法对绍兴市耳聋康复中心确诊的93例耳聋患儿,采用基质辅助激光解吸电离飞行时间质谱技术(MALDI-TOF-MS)检测4个常见耳聋基因GJB2、GJB3、MT-RNR1和SLC26A4的20个热点突变;采用Sanger测序法检测突变基因的全外显子,对Sanger测序无法解决的部分则采用长片段PCR、Gap PCR和定量PCR进行检测。结果 93例耳聋患儿经MALDI-TOF-MS检测,其中48例检出耳聋基因突变,检出率为51.61%。其中GJB2基因突变35例,检出率为37.63%,分别为致病突变24例,杂合突变11例;SLC26A4基因突变13例,检出率为13.98%,分别为SLC26A4致病突变6例,杂合突变7例;未检出MT-RNR1和GJB3基因突变。对18例检出杂合突变患儿中的17例进行突变杂合基因的全外显子检测,结果检出12例(70.59%)存在其他位点的杂合突变。最终42例患儿检出耳聋致病基因,检出率为45.16%。结论 93例耳聋患儿中,GJB2、SLC26A4基因检出率较高,对常规筛查中发现的携带杂合突变基因患儿进行该基因的全外显子检测,有助于提高耳聋致病基因检出率。     

菏泽市苯丙酮尿症患儿苯丙氨酸羟化酶基因突变特点

目的 分析菏泽市苯丙酮尿症(PKU)患儿苯丙氨酸羟化酶(PAH)基因突变特点,为深入开展PKU的诊断以及进一步的基因治疗提供科学依据。方法 采用高通量测序技术对临床确诊且自愿进行基因检测的PKU患儿的遗传代谢病相关基因进行检测,检出位点采用sanger测序进行验证及父母验证。结果 28个患者经测序分析后,共检出致病变异位点53个,总检出率为94.6%。其中25个患者检测到2个致病突变(89.3%),3个患者检测出1个突变位点(10.7%)。在53种变异类型中,错义突变38种(71.7%)、剪接突变4种(7.5%)、无义突变11种(20.8%)。变异位点分布在PAH基因第2、3、6、7、9~11外显子以及第4内含子区。外显子区变异位点50个(94.3%),内含子区变异位点3个(5.7%)。经典型PKU患儿以 c.1068C＞A为主,轻度PKU患儿以c.158G＞A、c.728G＞A为主,轻度高苯丙氨酸血症(HPA)以c.158G＞A及c.1068C＞A为主。c.158G＞A、c.728G＞A和c.1068c＞A的检出人数较多,c.158G＞A在轻度PKU及轻度HPA患儿中检出,平均Phe浓度为860.74 μmol/L,c.728G＞A在经典型PKU、轻度PKU患儿中检出,平均Phe浓度为879.51 μmol/L,c.1068C＞A在经典型PKU及轻度HPA患儿中检出,平均Phe浓度为1 098.44 μmol/L。结论 菏泽市PKU患儿PAH基因突变以c.158G＞A、c.728G＞A及c.1068C＞A突变为主。     

外显子测序技术在疾病研究中的应用

新一代测序技术的发展为疾病研究带来了新的机遇。在现阶段,外显子测序与全基因组测序相比,不仅费用更低,数据的阐释也更为简单。通过对疾病样本进行外显子组捕获测序,不仅能快速发现罕见遗传疾病的致病基因,也能用于研究多基因引起的常见疾病,如癌症、糖尿病、高血压等,来揭示这些疾病的遗传致病机理。外显子测序技术是当前的热点技术,极大地推动了疾病研究的进展。     

分子杂交技术在乙型肝炎拉米夫定治疗后YMDD变异检测中的应用

目的:采用分子杂交技术检测乙型肝炎拉米夫定治疗后发生的YMDD变异基因。方法:对100例服用拉米夫定12个月以上的乙型肝炎患者的血清,采用聚合酶链反应后进行分子杂交技术测定YMDD变异情况,同时采用乙肝P区基因测序技术进行结果比较。结果:100例拉米夫定治疗患者血清经分子杂交技术共检出33例YMDD变异,同测序结果吻合率达96.97%,灵敏度达103IU/ml。33例YMDD变异者共有6种不同的变异组合。结论:分子杂交技术检测HB-VYMDD变异具有简单、准确、快速、经济实用等特点,具有很好的临床应用价值。     

143例流产组织物的遗传学分析

目的:利用低覆盖度全基因组测序(WGS)结合荧光定量聚合酶链式反应(QF-PCR)技术检测早中孕期流产组织物,以明确其遗传学病因。方法:纳入143例早、中孕期流产组织物及孕妇外周血样本,用QF-PCR方法鉴定是否存在母源细胞污染、是否为三倍体;用WGS方法对阴性样本检测。结果:QF-PCR方法检出3例样本为100%母源样本污染,3例样本为三倍体,经荧光原位杂交(FISH)方法验证;WGS方法共检出53例异常,包括43例非整倍体异常,10例拷贝数变异(CNV)。结论:WGS结合QF-PCR技术用来检测流产组织物快速、可靠,有助于明确病因及进一步的生育指导。     

通过外显子组测序对1例孤独症谱系障碍患者的致病基因分析

目的 应用外显子测序技术对1例孤独症谱系障碍的男童进行外显子组测序分析,寻找致病基因。方法 选择1例就读于自闭症特殊学校的5岁男童,收集其临床资料,提取外周静脉血DNA,应用Nimblegen外显子序列捕获芯片及高通量测序技术对外显子区域进行测序。结果 通过全外显子组捕获测序发现了非同义突变11 309个,移码突变365个,同义突变23 075个,其中发现有4个基因的突变已有文献报道可能与孤独症谱系障碍的发病有关,这4个基因包括PTEN、AFF2、LAMB1、NRCAM。结论 应用外显子组捕获测序对该例孤独症谱系障碍的男童进行测序分析显示,大量单核苷酸多态性可能与该病的发生有关,也进一步证实了PTEN、AFF2、LAMB1、NRCAM这4个基因可能与孤独症谱系障碍的发生有关,有待进一步大样本研究及实验室研究验证其在孤独症谱系障碍发病中的具体机制。     

新型冠状病毒Alpha变异株实验室检测研究

目的 对一例新冠肺炎境外输入病例进行实验室检测,并对新型冠状病毒（severe acute respiratory syndrome coronavirus 2,SARS - CoV - 2）变异株进行鉴定,了解不同样本类型中新冠病毒的检出情况。方法 采集咽拭子、痰液、粪便、尿液和血液多种类型样本共13份,分别提取核酸,用荧光RT - PCR法检测SARS - CoV - 2。使用纳米孔MinION Mk1C测序仪,对阳性咽拭子样本进行实时SARS - CoV - 2靶向扩增全基因组测序,运用artic - ncov2019、DNAstar和Mega等软件对测定序列进行处理和分析。结果 咽拭子、痰液、粪便、血浆和尿沉渣样本中均检出SARS - CoV - 2核酸,咽拭子和血浆样本在发病的前四天均能检出,尿沉渣样本在发病当天能检出。基于纳米孔测序技术,7 h即获得SARS - CoV - 2全基因组数据,经分析显示为新冠病毒Alpha变异株。结论 本例境外输入新冠肺炎确诊病例的病原为SARS - CoV - 2 Alpha变异株,咽拭子、血浆样本和尿沉渣样本在发病早期均能检出SARS - CoV - 2核酸。     

Combining sequence data from multiple studies: Impact of analysis strategies on rare variant calling and association results

Individual sequencing studies often have limited sample sizes and so limited power to detect trait associations with rare variants. A common strategy is to aggregate data from multiple studies. For studying rare variants, jointly calling all samples together is the gold standard strategy but can be difficult to implement due to privacy restrictions and computational burden. Here, we compare joint calling to the alternative of single-study calling in terms of variant detection sensitivity and genotype accuracy as a function of sequencing coverage and assess their impact on downstream association analysis. To do so, we analyze deep-coverage (~82×) exome and low-coverage (~5×) genome sequence data on 2,250 individuals from the Genetics of Type 2 Diabetes study jointly and separately within five geographic cohorts. For rare single nucleotide variants (SNVs): (a) ≥97% of discovered SNVs are found by both calling strategies; (b) nonreference concordance with a set of highly accurate genotypes is ≥99% for both calling strategies; (c) meta-analysis has similar power to joint analysis in deep-coverage sequence data but can be less powerful in low-coverage sequence data. Given similar data processing and quality control steps, we recommend single-study calling as a viable alternative to joint calling for analyzing SNVs of all minor allele frequency in deep-coverage data.     

一种KRAS基因突变检测方法的建立及初步应用

目的建立一种KRAS基因实时荧光定量PCR-Sanger测序突变检测方法,并初步探讨其临床应用价值。方法以KRAS基因热点突变区域12、13密码子为研究位点设计特异性扩增、测序引物,利用已知野生型、突变型样品以TA克隆技术构建相应质粒作为标准品,建立KRAS基因实时荧光定量PCR-Sanger测序突变检测方法,并进行方法学和应用评估。结果成功构建了KRAS基因12、13密码子野生型、突变型质粒。建立了KRAS基因实时荧光定量PCR-Sanger测序突变检测方法,该方法灵敏度高(9.39×101copies/uL),重复性好(实时荧光定量PCR部分批内、批间变异系数分别为1.60%、2.54%)。该法与传统Sanger测序法同时检测40例临床样品,结果符合率为100%。结论本研究成功建立了可用于临床样品检测的KRAS基因实时荧光定量PCR-Sanger测序突变检测方法。     

Identifying Rare Variants With Optimal Depth of Coverage and Cost‐Effective Overlapping Pool Sequencing

Genome‐wide association studies have identified hundreds of genetic variants associated with complex diseases although most variants identified so far explain only a small proportion of heritability, suggesting that rare variants are responsible for missing heritability. Identification of rare variants through large‐scale resequencing becomes increasing important but still prohibitively expensive despite the rapid decline in the sequencing costs. Nevertheless, group testing based overlapping pool sequencing in which pooled rather than individual samples are sequenced will greatly reduces the efforts of sample preparation as well as the costs to screen for rare variants. Here, we proposed an overlapping pool sequencing to screen rare variants with optimal sequencing depth and a corresponding cost model. We formulated a model to compute the optimal depth for sufficient observations of variants in pooled sequencing. Utilizing shifted transversal design algorithm, appropriate parameters for overlapping pool sequencing could be selected to minimize cost and guarantee accuracy. Due to the mixing constraint and high depth for pooled sequencing, results showed that it was more cost‐effective to divide a large population into smaller blocks which were tested using optimized strategies independently. Finally, we conducted an experiment to screen variant carriers with frequency equaled 1%. With simulated pools and publicly available human exome sequencing data, the experiment achieved 99.93% accuracy. Utilizing overlapping pool sequencing, the cost for screening variant carriers with frequency equaled 1% in 200 diploid individuals dropped to at least 66% at which target sequencing region was set to 30 Mb.     

32例三阴性乳腺癌患者易感基因胚系突变分析

目的:应用全外显子测序技术初步探讨三阴性乳腺癌(TNBC)患者易感基因突变情况。方法:收集本院就诊的32例TNBC患者,均经临床手术病理确诊。采集患者外周血提取基因组DNA进行全外显子组测序,通过生物信息学分析筛选与乳腺肿瘤相关的易感基因变异。结果:32例TNBC患者中14例检测到BRCA1/2罕见变异,明确致病性或可疑致病变异6例,突变携带频率为18.8%。其中BRCA1:c.5468-1_5474del和c.4749_4750del是较常见的突变;BRCA2:c.6027A>C为新的变异;BRCA2:c.3794G>T、c.7901T>A,BRCA1:c.4616T>C首次在中国人群中发现。除了BRCA1/2变异外,还检测到83个乳腺肿瘤易感基因变异,每个患者携带2.6个变异。2个以上患者携带的乳腺癌易感基因包括ALK、APC、CDH1、PTCH2、RB1CC1、RAD51D、RAD54L、TSC1等。结论:BRCA1/2是TNBC患者最重要的易感基因,其他与DNA损伤修复相关的基因突变可能与TNBC患者的表型有一定的相关性。     

应用探针熔解分析法检测结核分枝杆菌rpoB基因突变

目的 研究结核分枝杆菌利福平耐药突变实时PCR检测试剂盒(探针熔解分析)的临床应用价值.方法 用37株非结核分枝杆菌考察该方法的特异性,用菌株H37Rv考察其检测限,并检测962份结核分枝杆菌培养标本的rpoB基因利福平耐药决定区突变,检测结果经测序验证.结果 37株非结核分枝杆菌中仅有3株出现耐药突变峰,检测限考察结果表明该方法每反应可重复检出30个菌.用该试剂盒检测962份标本,检出突变株186份,野生株751份,扩增失败标本25份.选取2009年11月以后的标本中试剂盒检出为突变的标本112份,并数字表法随机选取200份试剂盒检出为阴性的标本,进行测序验证,除5份测序失败其余107份标本的DNA序列分析结果与试剂盒检测结果一致.结论 该试剂盒准确快速,方便易行,是检测结核分枝杆菌rpoB基因突变的有力工具.

Abstract：

Objective To evaluate the clinical performance of a probe melting analysis ( PMA)-based real-time PCR detection kit in rapid detection of rifampin-resistant mutations in Mycobacterium tuberculosis (MTB). Methods The specificity of the assay was evaluated by detecting 37 non-tuberculous mycobacteria (NTM) ,and the detection limit of the method was evaluated by genomic DNA of a standard strain H37Rv. Finally, 962 clinical isolates were analyzed with the PMA assay by detecting mutations in rifampin resistance-determining region (RRDR) of rpoB gene, and results were verified with DNA sequencing. Results Among 37 NTM strains,three strains showed drug resistant mutation signals. The PMA method could detect down to 30 bacteria per reaction. Sample analysis showed that 186 of 962 isolates were mutants,751 isolates were wild type and 25 isolates failed to give amplification signals. Among the mutant samples detected, 112 samples from November 2009 to April 2010 were further analyzed by sequencing, as well as 200 wild-type samples. The results showed a complete agreement with the PMA assay except for 5 samples failed in sequence analysis. Conclusion The PMA assay is rapid, accurate and easy-to-use, and thus can be used for detection of rifampin-resistant in clinical isolate samples.     

乙型肝炎病毒"a"决定簇突变对乙型肝炎疫苗保护效果的影响

目的研究乙型肝炎（乙肝）病毒（HBV）“a”决定簇热点突变对乙肝疫苗保护效果的影响。方法根据HBV基因保守序列设计PCR扩增引物,针对“a”决定簇突变设计简并探针,制备检测HBV“a”决定簇16种热点突变的基因芯片,并用克隆测序对芯片检测结果进行验证。基因芯片法检测47对乙肝疫苗阻断失败的母婴配对样本和323例阻断成功的母亲样本。结果克隆测序证明,基因芯片法具有特异性。应用该法检测发现,野生株（阳性率为78．66%）仍为主要流行株,依次为126A（11．27%）、145R（5．76%）、126S-1（5．28%）、126S-2（4．56%）、129H（1．20%）、144A（0．72%）、129R（0．24%）,但126A阳性率显著高于其他突变（P值均＜0．01）。阻断失败组母婴均感染的样本与阻断成功组的126A、126S-1、126S-2和145R检出率的差异无统计学意义。结论现有乙肝疫苗可阻断126A、126S和145R突变株的母婴传播,目前毋需研制针对126和145位点突变的新型乙肝疫苗,但需加强对突变株的流行病学监测。     

On optimal pooling designs to identify rare variants through massive resequencing

The advent of next‐generation sequencing technologies has facilitated the detection of rare variants. Despite the significant cost reduction, sequencing cost is still high for large‐scale studies. In this article, we examine DNA pooling as a cost‐effective strategy for rare variant detection. We consider the optimal number of individuals in a DNA pool to detect an allele with a specific minor allele frequency (MAF) under a given coverage depth and detection threshold. We found that the optimal number of individuals in a pool is indifferent to the MAF at the same coverage depth and detection threshold. In addition, when the individual contributions to each pool are equal, the total number of individuals across different pools required in an optimal design to detect a variant with a desired power is similar at different coverage depths. When the contributions are more variable, more individuals tend to be needed for higher coverage depths. Our study provides general guidelines on using DNA pooling for more cost‐effective identifications of rare variants. Genet. Epidemiol. 35:139‐147, 2011. © 2011 Wiley‐Liss, Inc.     

Using Whole Exome Sequencing to Identify Candidate Genes With Rare Variants In Nonsyndromic Cleft Lip and Palate

Studies suggest that nonsyndromic cleft lip and palate (NSCLP) is polygenic with variable penetrance, presenting a challenge in identifying all causal genetic variants. Despite relatively high prevalence of NSCLP among Amerindian populations, no large whole exome sequencing (WES) studies have been completed in this population. Our goal was to identify candidate genes with rare genetic variants for NSCLP in a Honduran population using WES. WES was performed on two to four members of 27 multiplex Honduran families. Genetic variants with a minor allele frequency > 1% in reference databases were removed. Heterozygous variants consistent with dominant disease with incomplete penetrance were ascertained, and variants with predicted functional consequence were prioritized for analysis. Pedigree‐specific P‐values were calculated as the probability of all affected members in the pedigree being carriers, given that at least one is a carrier. Preliminary results identified 3,727 heterozygous rare variants; 1,282 were predicted to be functionally consequential. Twenty‐three genes had variants of interest in ≥3 families, where some genes had different variants in each family, giving a total of 50 variants. Variant validation via Sanger sequencing of the families and unrelated unaffected controls excluded variants that were sequencing errors or common variants not in databases, leaving four genes with candidate variants in ≥3 families. Of these, candidate variants in two genes consistently segregate with NSCLP as a dominant variant with incomplete penetrance: ACSS2 and PHYH. Rare variants found at the same gene in all affected individuals in several families are likely to be directly related to NSCLP.