转录组案例分享课件.ppt

1、1转录组案例分享转录组案例分享21 1、RNA-seq+small RNARNA-seq+small RNA2 2、DGE+small RNADGE+small RNA3 3、small RNA+small RNA+降解组测序降解组测序4 4、全转录组分析（、全转录组分析（mRNAmRNA，sRNAsRNA，LncRNALncRNA）1、转录组转录组 +RNA-seq/DGE+RNA-seq/DGE2 2、转录组、转录组+small RNA+small RNA3 3、转录组、转录组 +RNA-seq+RNA-seq/DGE/DGE+small RNA+small RNA4 4、转录组、转录组+

2、蛋白组蛋白组有参考基有参考基因组因组无参考基无参考基因组因组综合的研究策略综合的研究策略3Deep RNA sequencing at single base-pair resolution reveals high complexity of the rice transcriptomeGenome Res 2010案例案例 1 1水稻转录组水稻转录组实验材料 愈伤组织愈伤组织 根尖根尖（14d）茎尖茎尖（14d）叶叶（2 stages）稻花稻花/稻穗稻穗（3 stages）研究技术 转录组测序转录组测序（pair-end&single end）数字基因表达谱数字基因表达谱（DGE）smal

3、l RNA测序测序（18-30 nt）水稻基因注释水稻基因注释 水稻基因结构研究水稻基因结构研究 研究转录融合基因研究转录融合基因水稻转录组水稻转录组4鉴定新转录单元Case 1 Rice transcriptome sequencingNovel transcriptomeNovel miRNAFeature of novel transcriptome5水稻的可变剪接水稻的可变剪接33%of the rice genes exist alternative splicing events6水稻的可变剪接水稻的可变剪接IR is the main kind of AS events,whic

4、h is unlike human(ES is the main kind of AS events).7前列腺癌转录组案例案例 2 21.14 primary prostate cancers and their paired normal counterparts from the Chinese population using RNA-seq2.Three of the 14 tumors(21.4%)harbored a TMPRSS2-ERG fusion3.Two novel gene fusions,CTAGE5-KHDRBS3 and USP9Y-TTTY15,occurre

5、d frequently in our patient cohort4.The correlation between expression of long ncRNA and genes suggested that long ncRNAs may have functions beyond transcriptional regulation8研究策略14 matched pairs of human primary prostate cancer and adjacent tissues RNA re-sequencing and Long ncRNA sequencingMutatio

6、nIntergrative Pathway Analysis Gene Expression Gene FusionLong Non-coding RNA Alternative Splicing9融合基因研究Previous known gene fusionTMPRSS2-ERG10融合基因研究Frequent novel gene fusionUSP9Y-TTTY1511长链非编码RNA研究Supervised hierarchical clustering analysis of 137 long ncRNAsLong non-coding RNA12长链非编码RNA研究Long no

7、n-coding RNA13红花转录组测序案例案例 3 3De Novo Transcriptome of Safflower and the Identification of Putative Genes for Oleosin and theBiosynthesis of FlavonoidsSafflower(Carthamus tinctorius L.)is one of the most extensively used oil crops in the world.However,little is known about how its compounds are synth

8、esized at the genetic level.In this study,Solexa-based deep sequencing on seed,leaf and petalof safflower produced a de novo transcriptome consisting of 153,769 unigenes.We annotated 82,916 of the unigenes with gene annotation and assigned functional terms and specific pathways to a subset of them.M

9、etabolic pathway analysis revealed that 23 unigenes were predicted to be responsible for the biosynthesis of flavonoids and 8 were characterized as seed-specific oleosins.In addition,a large number of differentially expressed unigenes,for example,those annotated as participating in anthocyanin and c

10、halcone synthesis,were predicted to be involved in flavonoid biosynthesis pathways.In conclusion,the de novo transcriptome investigation of the unique transcripts provided candidate gene resources for studying oleosin-coding genes and for investigating genes related to flavonoid biosynthesis and met

11、abolism in safflower.14背景介绍：背景介绍：主要的油料作物之一 红花种子油中含有较高的亚油酸，有降低血脂及血清胆固醇、软化和扩张动脉、防止动脉粥样硬化 红花花瓣中含有黄酮类化合物有抗氧化作用 没有参考基因组信息 遗传水平上的化合物合成机制不清楚研究目的：研究目的：丰富现有红花转录本数据库信息 探寻与油质蛋白相关的基因 探寻与黄酮类化合物合成相关的基因红花转录组测序15红花转录组测序材料选取：材料选取：种子，叶，花瓣三个组织各一个样品 各20ug Total RNA样品方法思路：方法思路：样本：红花三个不同组织个1个样品技术方法：Illumina GA II 75PE 测序

12、分析方法：denovo从头组装，差异分析以及KEGG，GO注释1.验证：RT-PCR验证16红花转录组测序信息分析方法：信息分析方法：建库建库（200bp）Illumina GAII测序测序SOAPdenovoSOAPdenovo组装组装组装结果组装结果NrNr，COGCOG，GOGO，KEGGKEGG注释注释不同组织间差异不同组织间差异表达基因筛选表达基因筛选GOGO，pathwaypathway富集富集分析分析差异基因差异基因RT-PCRRT-PCR验证验证17红花转录组测序结果一：结果一：基本数据统计18红花转录组测序结果二：结果二：WEGO分析19红花转录组测序结果三：结果三：黄酮素生

13、物合成相关的pathway分析20红花转录组测序结果四：结果四：RT-PCR挑选3个差异基因进行验证，其中12个与测序结果趋势一致21红花转录组测序结论：结论：u A total of 153,769 unigenes were generated by Illumina Solexa de novo sequencing technologyu 23 candidate unigenes from the petal cDNA library were characterized to be responsible for the biosynthesis of flavonoidsu 8

14、unigenes were putatively characterized as seed-specific oleosinsu The de novo transcriptome could be useful and cost-effective for distinguishing transcripts,functional genes and for providing quantitative estimates of gene expression22飞蝗转录组测序案例案例 4 4De Novo Analysis of Transcriptome Dynamics in the

15、 Migratory Locust during the Development of Phase TraitsLocusts exhibit remarkable density-dependent phenotype(phase)changes from the solitary to the gregarious,making them one of the most destructive agricultural pests.This phenotype polyphenism arises from a single genome and diverse transcriptome

16、s in different conditions.Here we report a de novo transcriptome for the migratory locust and a comprehensive,representative core gene set.We carried out assembly of 21.5 Gb Illumina reads,generated 72,977 transcripts with N50 2,275 bp and identified 11,490 locust protein-coding genes.Comparative ge

17、nomics analysis with eight other sequenced insects was carried out to indentify the genomic divergence between hemimetabolous and holometabolous insects for the first time and 18 genes relevant to development was found.We further utilized the quantitative feature of RNA-seq to measure and compare ge

18、ne expression among libraries.We first discovered how divergence in gene expression between two phases progresses as locusts develop and identified 242 transcripts as candidates for phase marker genes.23飞蝗转录组测序背景介绍：背景介绍：主要的农业害虫之一，导致巨大的经济和生态破坏 重要生理学模型，对外界刺激会有表型上的改变 有散居型和群居型两种表型 基因组大小约为6.5G，目前还没有参考基因组

19、信息 目前数据库中有一些EST序列，但是没有新一代测序数据研究目的：研究目的：丰富飞蝗转录组信息，找到飞蝗关键基因集 探寻飞蝗不同表型的标志基因 探寻飞蝗发育相关基因24飞蝗转录组测序材料选取：材料选取：两个不同表型：散居型和群居型 每个表型不同发育阶段各一个样品：egg,combined 1st and 2nd instar,3rd instar,4th instar,5th instar,and adult方法思路：方法思路：样本：两个表型，每个表型6个发育时间点共12个文库技术方法：Illumina GA II 测序（PE and SE）分析方法：denovo从头组装，基因表达量分析以及

20、KEGG，GO注释1.验证：RT-PCR验证25飞蝗转录组测序信息分析方法：信息分析方法：转录建库转录建库（200bp）Illumina GAII PE 测序测序SOAPdenovoSOAPdenovo组装组装组装结果注组装结果注释释不同组织间差异不同组织间差异表达基因筛选表达基因筛选GOGO，pathwaypathway富集分析富集分析RT-PCRRT-PCR验证验证数字基因表达谱数字基因表达谱建库建库（17bp）Illumina GAII SE 测序测序基因定量分基因定量分析析reference26飞蝗转录组测序结果一结果一：（不同组装方式以及：（不同组装方式以及K-mer值探寻）值探寻）

21、27飞蝗转录组测序结果二：结果二：PCAPCA分析发现不分析发现不同发育阶段影响同发育阶段影响要比两种表型影要比两种表型影响大响大28飞蝗转录组测序结果三：结果三：两种表型之间相同发两种表型之间相同发育阶段比较，说明在育阶段比较，说明在4 4龄期，两者发育发生龄期，两者发育发生巨大变化，在以后各巨大变化，在以后各时期维持这种变化时期维持这种变化29飞蝗转录组测序结果四：结果四：GOGO和和KEGGKEGG富集分析，富集分析，发现发现4 4龄期与龄期与5 5龄期龄期和成虫期功能正好和成虫期功能正好相反相反30飞蝗转录组测序结论：结论：u 对于Denovo组装，不同样品先分开组装，然后聚类效果比把

22、不同样品reads合并再组装最后聚类效果要好u 本次研究得到飞蝗72,977个转录本，鉴定到11,490飞蝗编码蛋白基因u 鉴定到242个候选表型标记基因，其中群居型在环境信息发现与处理Pathway中基因表达活跃；散居型在新陈代谢和生物合成Pathway中基因表达活跃31心力衰竭全转录组案例案例 5 51.研究目的研究目的为了更全面的研究心力衰竭以及辅助装置下重塑过程相关的心肌细胞动态变化2.实验设计实验设计非缺血性心力衰竭植入辅助装置前后的心肌细胞样品8对，缺血性性心力衰竭植入辅助装置前后的心肌细胞样品8对，正常心肌细胞样品8个。分别对40个样品进行RNA-seq，lncRNA-seq，m

23、iRNA-seq测序 3.结论结论心肌转录组分析发现lncRNA能够明显区分心力衰竭的不同类型，而且对植入辅助装置前后的变化比较灵敏，本研究认为，lncRNA在心力衰竭的不同类型以及植入辅助装置后的重塑中起到重要作用。32初步数据统计初步数据统计NF：正常样品，ICM：缺血性，NICM：非缺血性，pre-LVAD：植入辅助装置前，post-LVAD:植入辅助装置后RNAseq：89%reads比对上基因组hg19,56.6%比对上外显子，3.9%比对上内含子，32.8%比对上基因间区。miRNAseq：97.7%reads唯一比对上基因组，66.1%比对上已知miRNA，14.1%比对上其他小RNA。33分析流程分析流程34lncRNA能够更好的区分ICM和NICM35 Expression of Cardiac lncRNAs and Neighboring Coding Genes Is Highly CorrelatedRARA：retinoic acid receptor alpha 36 lncRNA s to combined mRNA and miRNA expression profiles increases the power to discriminate NICM samp les before and after LVAD support37