外显子组测序介绍


外显子组测序(Exomesequencing)是指利用序列捕获技术将全基因组外显子区域DNA捕捉并富集后进行高通量测序的基因组分析方法。外显子组是指全部外显子区域的集合,该区域包含合成蛋白质所需要的重要信息,涵盖了与个体表型相关的大部分功能性变异。由于外显子组测序只需针对外显子区域的DNA即可(例如:人类外显子组只占人类基因组长度的约1%,但是目前由DNA变异引起的疾病估计有85%以上来自于外显子组区域的变异),因此远比进行全基因组序列测序更简便、经济、高效,其目标区域覆盖度也更高,便于变异检测。该测序可用于检测外显子DNA中的单核苷酸多态性,插入缺失,结构变化,拷贝数变化等,预测个体水平或者群体水平的基因表达差异性比较,也常用于临床上进行疾病相关基因信息获取,用于临床指导个性化治疗方案的制定。

目前,外显子测序已经在米勒综合症、歌舞伎综合症、重型颅脑畸形等孟德尔疾病的研究中得到成功应用。还有其它一些癌症和复杂性疾病也应用外显子测序观察到高度相关的突变。可用于寻找复杂疾病(如:癌症、糖尿病、肥胖症等)的致病基因和易感基因等的研究。


应用案例:

Exome Sequencing Identifies DLG1 as a Novel Gene for Potential Susceptibility to Crohn's Disease in a Chinese Family Study


Exome sequence data from 4 individuals belonging to a same family were analyzed using bioinformatics methods to narrow down the variants associated with CD. The potential risk genes were further analyzed by genotyping and Sanger sequencing in family members, additional 401 healthy controls (HC), 278 sporadic CD patients, 123 UC cases, a pair of monozygotic CD twins and another Chinese CD family.

Results


From the CD family in which the father and daughter were affected, we identified a novel single nucleotide variant (SNV) c.374T>C (p.I125T) in exon 4 of discs large homolog 1 (DLG1), a gene has been reported to play mutiple roles in cell proliferation, T cell polarity and T cell receptor signaling. After genotyping among case and controls, a PLINK analysis showed the variant was of significance (P<0.05). 4 CD patients of the other Chinese family bore another non-synonymous variant c.833G>A (p.R278Q) in exon 9 of DLG1.


Conclusions

We have discovered novel genetic variants in the coding regions of DLG1gene, the results support that DLG1 is a novel potential susceptibility gene for CD in Chinese patien

 

Chromatogram of DLG1 gene mutations.

The Sanger sequence traces from normal human controls are shown in panel A and B; the mutations were heterozygous at the corresponding locus (orange arrows indicating) in panel C and D.


外显子测序对DNA样品的要求如下:

样品类型:(一)无降解且无RNA污染的DNA样品; (二)样品的量(单次):≥ 3 µg(使用Agilent 液相平台); (三)样品浓度:≥ 50 ng/µl (使用Agilent 液相平台); (四)样品纯度:OD260/280= 1.8~2.0。外显子测序后获得原始数据(raw reads),经过过滤去污染、比对参考基因组,获得比对到基因组上的Unique mapped reads。对数据进行标准的信息分析流程,包括对SNP、InDel等进行检测、注释和统计分析。同时对数据进行质控检测,包括测序深度、覆盖度均一性等分析的检测报告。

生物信息学分析:

外显子测序后获得原始数据(raw reads),经过过滤去污染、比对参考基因组,获得比对到基因组上的Unique mapped reads。对数据进行标准的信息分析流程,包括对SNP、InDel等进行检测、注释和统计分析。同时对数据进行质控检测,包括测序深度、覆盖度均一性等分析。


标准信息分析

数据产出的统计

外显子区域测序深度的直方分布图

外显子捕获的均一性分析

外显子融合分析

一致性序列的获得和SNPs的检测SNPs的注释

插入和缺失(Indels)的检测

插入和缺失(Indels)的注释

个性化分析

氨基酸替代的预测分析

群体SNP的获取和等位基因频率评估

Mendelian disorder analysis 孟德尔遗传疾病分析

基于新一代测序技术的全基因组关联(NGS-GWAS)分析

基因融合


已有发表的相关文献:

(1)外显子组测序

Diagnostic clinical genome and exome sequencing.

Biesecker LG, Green RC.

N Engl J Med. 2014 Jun 19;370(25):2418-25. doi: 10.1056/NEJMra1312543. No abstract available.


IF:51.658


Exome sequencing in unspecific intellectual disability and rare disorders.

Rauch A.

Mol Cytogenet. 2014 Jan 21;7(Suppl 1 Proceedings of the International Conference on Human):I26. doi: 10.1186/1755-8166-7-S1-I26. eCollection 2014. No abstract available.

IF:2.36


Mutations of 60 known causative genes in 157 families with retinitis pigmentosa based on exome sequencing.

Xu Y, Guan L, Shen T, Zhang J, Xiao X, Jiang H, Li S, Yang J, Jia X, Yin Y, Guo X, Wang J, Zhang Q.

Hum Genet. 2014 Jun 18. [Epub ahead of print]

IF:10.603


Exome Sequencing Identifies DLG1 as a Novel Gene for Potential Susceptibility to Crohn's Disease in a Chinese Family Study.

Xu S, Zhou F, Tao J, Song L, Ng SC, Wang X, Chen L, Yi F, Ran Z, Zhou R, Xia B.

PLoS One. 2014 Jun 17;9(6):e99807. doi: 10.1371/journal.pone.0099807. eCollection 2014.

           

IF:3.05



Whole exome sequencing identifies a novel splice-site mutation in ADAMTS17 in an Indian family with Weill-Marchesani syndrome.

Shah MH, Bhat V, Shetty JS, Kumar A.

Mol Vis. 2014 Jun 12;20:790-6. eCollection 2014.

IF:2.205


Exome Sequencing Reveals the Likely Involvement of SOX10 in Uveal Melanoma.

Das D, Kaur I, Ali MJ, Biswas NK, Das S, Kumar S, Honavar SG, Maitra A, Chakrabarti S, Majumder PP.

Optom Vis Sci. 2014 Jun 12. [Epub ahead of print]

IF: 3.441


Whole exome sequencing identifies new causative mutations in tunisian families with non-syndromic deafness.

Riahi Z, Bonnet C, Zainine R, Louha M, Bouyacoub Y, Laroussi N, Chargui M, Kefi R, Jonard L, Dorboz I, Hardelin JP, Salah SB, Levilliers J, Weil D, McElreavey K, Boespflug OT, Besbes G, Abdelhak S, Petit C.

PLoS One. 2014 Jun 13;9(6):e99797. doi: 10.1371/journal.pone.0099797. eCollection 2014.

IF: 3..05

Investigation of pathogenic genes in peri-implantitis from implant clustering failure patients: a whole-exome sequencing pilot study.

Lee S, Kim JY, Hwang J, Kim S, Lee JH, Han DH.

PLoS One. 2014 Jun 12;9(6):e99360. doi: 10.1371/journal.pone.0099360. eCollection 2014.

IF: 3..05


Exome sequencing reveals CHM mutations in six families with atypical choroideremia initially diagnosed as retinitis pigmentosa.

Li S, Guan L, Fang S, Jiang H, Xiao X, Yang J, Wang P, Yin Y, Guo X, Wang J, Zhang J, Zhang Q.

Int J Mol Med. 2014 Jun 6. doi: 10.3892/ijmm.2014.1797. [Epub ahead of print]

IF:1.96


Exome sequencing of hepatoblastoma reveals novel mutations and cancer genes in the Wnt pathway and ubiquitin ligase complex.

Jia D, Dong R, Jing Y, Xu D, Wang Q, Chen L, Li Q, Huang Y, Zhang Y, Zhang Z, Liu L, Zheng S, Xia Q, Wang H, Dong K, He X.

Hepatology. 2014 Jun 9. doi: 10.1002/hep.27243. [Epub ahead of print]

IF:9.858


The usefulness of whole-exome sequencing in routine clinical practice.

Iglesias A, Anyane-Yeboa K, Wynn J, Wilson A, Truitt Cho M, Guzman E, Sisson R, Egan C, Chung WK.

Genet Med. 2014 Jun 5. doi: 10.1038/gim.2014.58. [Epub ahead of print]

IF:5.56


Whole exome sequencing reveals concomitant mutations of multiple FA genes in individual Fanconi anemia patients.

Chang L, Yuan W, Zeng H, Zhou Q, Wei W, Zhou J, Li M, Wang X, Xu M, Yang F, Yang Y, Cheng T, Zhu X.

BMC Med Genomics. 2014 May 15;7(1):24. doi: 10.1186/1755-8794-7-24.

IF:3.47


Exome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomas.

Zhang L, Chen LH, Wan H, Yang R, Wang Z, Feng J, Yang S, Jones S, Wang S, Zhou W, Zhu H, Killela PJ, Zhang J, Wu Z, Li G, Hao S, Wang Y, Webb JB, Friedman HS, Friedman AH, McLendon RE, He Y, Reitman ZJ, Bigner DD, Yan H.

Nat Genet. 2014 Jun 1. doi: 10.1038/ng.2995. [Epub ahead of print]


IF:35.53