基因組學課件：Transcritomics20131204

1、Understanding The Complexity of Transcriptomics,Jun YU BIG, CAS,2020/8/27,BIG-CAS,1,The transcripts of a genome,2020/8/27,BIG-CAS,2,mRNAs and their variants (90%) lncRNAs (antisense or sense) Small RNAs miRNAs Catalytic RNAs Other defined small RNAs (such as piRNAs) Not-yet-defined small RNAs RNAs f

2、rom BDRs (TEs),RNA Modifications,By types of modification: 107 By species of RNAs tRNA (92) rRNA (30) mRNA (13) tmRNA (two) snRNA (11) chromosomal RNA (1) other RNAs (1),2020/8/27,BIG-CAS,3,15,318,5,260,2,040,rmRNA-Seq,mRNA-Seq,(20,578),(17,358),Total Unique Reads,Total Protein Coding,15,809,723,550

3、,rmRNA-Seq,mRNA-Seq,(16,532),(16,359),2020/8/27,BIG-CAS,4,Annotated ncRNA,2020/8/27,BIG-CAS,5,Classification of Annotated ncRNA,Novel ncRNA,1,454,8,733,3,553,rmRNA-Seq,mRNA-Seq,(10,187),(5,007),2020/8/27,BIG-CAS,6,Gene coverage bias,2020/8/27,BIG-CAS,7,Methods to study transcriptomics,2020/8/27,BIG-

4、CAS,8,Hybridization-based Northern blotting FISH Microarrays Sequence-based ESTs: 1 to 10,000 Short tags: 100,000 million NGS-tags: 20 million per sampling,Meta-analysis for transcriptome,2020/8/27,BIG-CAS,9,Functional categorizations: HK or TS Pathways involved, such as circadian-regulated? Positio

5、ning on chromosome, such as cluster-regulated? Quantity for the number of sizes, copies and variants Tempospatial distribution for where it and its variants are actually expressed Is it really expressing: Antisense transcript? Chromosome status? miRNA regulation? Homeostatic controls?,2020/8/27,Jun

6、Yu. (2012). Life on Two Tracks. Genomics Proteomics Bioinf, online June 22, 2012.,BIG-CAS,10,Influences from Both EpiG and RiboG,Genotype,RiboG,EpiG,2020/8/27,BIG-CAS,A ribogenomic flip,A Epigenomic flpp,11,Env J,Env I,Genotype,Phenotype,Genome,Transcriptome,Proteome,Metabolome,Epigenome,Ribogenome,

7、Nucleosome-space occupancy,Histone Marks,Gene Clustering,mRNA Splicing,miRNAs,lncRNAs,Other Small RNAs,DNA modifications,O_Epigenome,O_Ribogenome,Chromatin Dynamics,Chromosome Folding,RNA Degradation,CpG density,2020/8/27,Physiome,Exposome,RNA Editing,RNA Routing,RNA Modification,BIG-CAS,12,13,2020/

8、8/27,RNA研究研討,基因組生物學之“五行”,信息流：遺傳學與變演論The Informational Track: Genetics and Evolution 基因組結構與變演Genomics and Comparative Genomics 基因型與表型界定Genotype -phenotype Relationship 操作流：細胞生物學與分子生物學 操作流之一：表觀組學The Operational Track-I: Epigenomics 操作流之二：核酸組學The Operational Track-II: Ribogenomics 操作流之三：蛋白質組學The Operat

9、ional Track-III: Proteomics 操作流之四：亞細胞器學 The Operational Track-IV: Organelles 平衡流：生物化學、生理學與藥理學 平衡流之一：能源流The Homeostatic Track-I: Energy 平衡流之二：物質流The Homeostatic Track-II: Matter 平衡流之三：信導流The Homeostatic Track-III: Signaling 分室流：生命的起源、系統(tǒng)發(fā)育和發(fā)生 分室流之一：生命起源The Compartmental Track-I： Origin of life 分室流之二：生

10、命發(fā)生 The Compartmental Track-II: Development of diverse lifeforms 分室流之三：細胞分化 The Compartmental Track-III：Differentiation of cells 可塑流：表型可塑性與神經生物學 可塑流之一：表型可塑性The Plasticity Track-I: Environmental Influence and Phenotypic Plasticity 可塑流之二：行為與認知的可塑性The Plasticity Track-II: The Plasticity of Affect, Beha

11、vior, and Cognition,2020/8/27,fudan Lecture,14,The Rubiks Cube Logic: the New Paradigm,Genes are regulated in such a way where,Puzzle-solving Rule-learning Discovery-driven,Mechanism-scrutinizing Learning relationships Hypothesis-driven,2020/8/27,RNA研究研討,15,The Complex View of Life,Puzzle-solving Ru

12、le-learning Discovery-driven,Epigenome,Genome,Proteome,Ribogenome,Homeostasome,Physiome,2020/8/27,RNA研究研討,16,Therapeutic Targeting of the Hallmarks of Cancer,2020/8/27,RNA研究研討,17,Source: Cell , Volume 144, Issue 5, Pages 646-674 (DOI:10.1016/j.cell.2011.02.013),Complex Mechanisms Lead to Clonal Hete

13、rogeneity,2020/8/27,18,RNA研究研討,2020/8/27,BIG-CAS,19,Lineage-based Characterization of Genomes,2020/8/27,20,RNA研究研討,2020/8/27,RNA研究研討,21,Different Splicing Mechanisms Determine Structures of All Genomes,2020/8/27,RNA研究研討,22,Splicing-1: half (small) introns, 25 bp Splicing-2: “minimal” introns, 100 bp

14、 Splicing-3: plant introns, 200 but 30 kbp,Two-track Biology May not be Not Enough,2020/8/27,RNA研究研討,23,Alarmones (pppGpp, AppppA, ApppG, etc.) Siderophores (such as enterochelin and vibriobactin) Heme Vitamin C,The 2+N-track Biology,Evolution at Two Levels in Humans and Chimpanzees Their Macromolec

15、ules Are So Alike That Regulatory Mutations May Account For Their Biological Differences.Mary-Claire King and Allan C. Wilson,2020/8/27,BIG-CAS,24,2020/8/27,BIG-CAS,25,Phenotype VariabilityPenetrance and Expressivity,A specific example of incomplete penetrance is the human bone disease osteogenesis

16、imperfecta (OI). The majority of people with this disease have a dominant mutation in one of the two genes that produce type 1 collagen, COL1A1 or COL1A2. Collagen is a tissue that strengthens bones and muscles and multiple body tissues. People with OI have weak bones, bluish color in the whites of

17、their eyes, and a variety of afflictions that cause weakness in their joints and teeth. However, this disease doesnt affect everyone who has COLIA1 and COLIA2 mutations in the same way. In fact, some people can carry the mutation but have no symptoms. Thus, families can unknowingly transmit the muta

18、tion from one generation to the next through someone who carries the mutation but does not express the OI phenotype. Miko, I. (2008) Phenotype variability: penetrance and expressivity. Nature Education 1(1),2020/8/27,BIG-CAS,26,Penetrance,Interestingly, some scientists have actually tried to do this

19、 by observing how the genetic mutations that cause OI affect mice. These investigators inserted a mutated form of COL1A1 into mice and bred them so that they all contained this mutation. The mice were affected in similar ways to those with human OI: Many had severe bone weakness and multiple bone fr

20、actures, even at birth. In fact, when the researchers examined the mouse bones closely, they found that 70% of mice with the mutated COL1A1 gene showed evidence of OI (bone fractures); however, the remaining 30% appeared completely normal. In these mice with no OI phenotype, there was the same amoun

21、t of COL1A1 expression as in those mice that did show the phenotype. Furthermore, the investigators used a purebred strain of mice that had little variability in their genomes to begin with. This means that the genetic context in which COL1A1 was expressed did not vary among the mice studied. Yet, d

22、espite the fact that all the mice had extremely similar genomes and all of them expressed the same amount of COL1A1, 30% of them did not show any OI phenotype. These results continue to be perplexing. Miko, I. (2008) Phenotype variability: penetrance and expressivity. Nature Education 1(1),2020/8/27

23、,BIG-CAS,27,An example of expressivity at work is the occurrence of extra toes, or polydactyly, in cats. The presence of extra toes on a cats paw is a phenotype that emerges in groups of cats who have interbred for generations. In fact, there are several well-known groups of these cats, such as thos

24、e on Key West Island (known as Hemingways cats), as well as those in breeding clusters in the eastern U.S. and shores of the British Isles (Figure 2). The first to report on this phenomenon was C. H. Danforth, who studied the inheritance of polydactyly among 55 generations of cats. He observed that

25、the polydactyly phenotype showed good penetrance, but variable expression because the gene always causes extra toes on the paw, but the number of extra toes varies widely from cat to cat (Danforth, 1947). Through his breeding studies, Danforth found that although a dominant allele underlies the caus

26、e of polydactyly, the degree of polydactyly depends on the condition of adjacent layered tissues in the developing limb; that is, the expression of genes in tissues surrounding tissue that will become the toe determines the degree of polydactyly (Willier, 1974).,2020/8/27,BIG-CAS,28,Wiki-definition,

27、Penetrance in genetics is the proportion of individuals carrying a particular variant of a gene (allele or genotype) that also express an associated trait (phenotype). In medical genetics, the penetrance of a disease-causing mutation is the proportion of individuals with the mutation who exhibit cli

28、nical symptoms. Highly penetrant alleles, and highly heritable symptoms, are easier to demonstrate, because if the allele is present, the phenotype is generally expressed. Mendelian genetic concepts such as recessiveness, dominance, and co-dominance are fairly simple additions to this principle.,202

29、0/8/27,BIG-CAS,29,Challenges to the Common Dogma,When genotypes do not match phenotypes: are we able to fit everything to the framework of genetics and evolution? “In biology, and specifically genetics, epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by

30、mechanisms other than changes in the underlying DNA sequence hence the name epi- (Greek: - over, above, outer) -genetics. from wikipedia” Too complex to fit in the current paradigm: DNA-centric, RNA-centric, protein-centric, metabolite-centric and compartment-centric mechanisms. New concepts, new te

31、chnology, and new data lead to new paradigm.,2020/8/27,BIG-CAS,30,Genetic Heterogeneity and Pleiotropy,Genetic Heterogeneity is a phenomenon in which a single phenotype or genetic disorder may be caused by any one of a multiple number of alleles or non-allele (locus) mutations.1 This is in contrast

32、to pleiotropy, where a single gene may cause multiple phenotypic expressions or disorders. Genetic heterogeneity can be classified as either allelic or locus. Pleiotropy occurs when one gene influences multiple phenotypic traits. Consequently, a mutation in a pleiotropic gene may have an effect on s

33、ome or all traits simultaneously. This can become a problem when selection on one trait favors one specific version of the gene (allele), while the selection on the other trait favors another allele.,2020/8/27,BIG-CAS,31,2020/8/27,32,BIG-CAS,2020/8/27,BIG-CAS,33,2020/8/27,34,BIG-CAS,DNA Modification

34、: 5 different Types,2020/8/27,BIG-CAS,35,2020/8/27,BIG-CAS,36,2020/8/27,BIG-CAS,37,Based on conventional natural selection, we hypothesized that the channels genes would have evolved mutations to help tune them to their respective environments. Surprisingly, the primary sequences encoded by the two

35、genes were virtually identical, differing at only four positions. A clear advantage to this strategy is that it allows options: different isoforms can be expressed in response to different conditions. Exactly how organisms exercise these options is largely unknown.,What We Have Learnt So Far (II),Ge

36、ne structural features are not randomly distributed among genomes. Such as Promoter sharing Minimal introns are not junk and may be functionally (known to be negatively selected) selected. Minimal introns may play functional roles in transcript exports.,2020/8/27,BIG-CAS,38,Distribution of minimal i

37、ntrons in human genes is non-random,2020/8/27,BIG-CAS,39,“polymorphism is just a transient phase of molecular evolution”Mooto Kimura,QUESTION: what would we find if we re-sequenced 93 minimal introns in an ethnically-diverse human population of 45.7 samples (or 91.4 chromosomes),2020/8/27,BIG-CAS,40

38、,2020/8/27,BIG-CAS,41,Why do “minimal introns” persist despite TE- and MS-bombardment?,BUT this cannot be the complete explanation,selection against too-small introns,mutational bias for deletions over insertions,2020/8/27,BIG-CAS,42,2020/8/27,BIG-CAS,43,2020/8/27,BIG-CAS,44,“Minimal Intron” Polymor

39、phisms,2020/8/27,BIG-CAS,45,2020/8/27,BIG-CAS,46,2020/8/27,BIG-CAS,Jun Yu. (2012). Challenges to the common dogma. Genomics Proteomics Bioinf 10(2):5557, online June 8, 2012.,47,Basic Data,2020/8/27,48,BIG-CAS,Dapeng Wang and Jun Yu. (2011) Both size and GC-content of minimal introns are selected in

40、 human population. PLoS One 6: e17945.,2020/8/27,49,BIG-CAS,DAF 0.1,RDI (DAF0.1) in different intron size intervals from combined datasets of the three populations.,2020/8/27,50,BIG-CAS,RDI: Ratio of Deletion over Insertion,GC effect: 65% more deletions,RDI (DAF0.1) in different intron size interval

41、s of the three populations,2020/8/27,51,BIG-CAS,2020/8/27,52,BIG-CAS,Table 3RDI (DAF0.1) under variable size intervals and GC-contents,2020/8/27,53,BIG-CAS,Median GC contents in variable intron size intervals and regions.,*We pooled both 5-exon and 3-exon data as flanking exon data and compared them

42、 with intron data. The P values were calculated based on Wilcoxon rank sum test.,2020/8/27,54,BIG-CAS,RDI (DAF0.1) when examining dependence of GC-effect on intron length.,2020/8/27,55,BIG-CAS,RDI (DAF0.1) when examining dependence of size-effect on GC contents.,*These data points are not in agreeme

43、nt with the size-effect.,2020/8/27,56,BIG-CAS,2020/8/27,57,BIG-CAS,2020/8/27,BIG-CAS,58,2020/8/27,BIG-CAS,59,2020/8/27,BIG-CAS,60,2020/8/27,BIG-CAS,61,2020/8/27,BIG-CAS,62,2020/8/27,BIG-CAS,63,Organizational Dynamicsof Genes and Chromosomes,2020/8/27,BIG-CAS,64,What Have We Learnt So Far (III),Mamma

44、ls have 21,000 genes and nearly 9,000 are house-keeping (HK); Sampling 15 m tags are good enough to acquire a transcriptome for a mammalian cell. House-keeping does not indicate expression levels: 700 genes are expression-invariable genes (EIGs) and others are EVGs. Highly expressed EIGs accumulates

45、 more mutations and the fact is true for all HK genes.,2020/8/27,BIG-CAS,65,Conservation Pattern of Gene Pairs：Divergent Gene Pairs （）,2020/8/27,BIG-CAS,66,Distance Between Genes,2020/8/27,BIG-CAS,67,Correlated Gene Expression,2020/8/27,BIG-CAS,68,Vertebrate DPGs and CPGs are more conserved,Yang L a

46、nd Yu J (2009) A Comparative Analysis of Divergently-Paired Genes (DPGs) of Drosophila and Vertebrate Genomes. BMC Evolutionary Biology 9:55.,2020/8/27,BIG-CAS,69,Evolution of DPGs in Insects and Vertebrates,2020/8/27,BIG-CAS,70,The statistics of nucleotide frequencies around transcriptional start s

47、ites (TSSs),Indicating tissue-specificity,2020/8/27,BIG-CAS,71,What Material To Use:A Unicellular Organism, Trichomonas vaginalis,Recently-duplicated genome Unicellular eukaryotes Mostly intronless genes Short UTRs Both genome and EST sequences available,2020/8/27,BIG-CAS,72,Power Spectrum Analysis,

48、2020/8/27,BIG-CAS,73,Aligned coding sequences,All sequences,周期性,2020/8/27,BIG-CAS,74,周期性 與GC梯度,2020/8/27,BIG-CAS,75,2020/8/27,BIG-CAS,76,2020/8/27,BIG-CAS,77,2020/8/27,BIG-CAS,78,2020/8/27,BIG-CAS,79,Regulatory dynamics: genes, gene products, and their interaction networks in cells,2020/8/27,BIG-CAS

49、,80,Human Transcriptomes,A single Cell,300 cell types,30 tissue types,Millions of SNPs and CNVs,1013 Cells,100,000 genes,2020/8/27,BIG-CAS,81,2020/8/27,BIG-CAS,82,2020/8/27,BIG-CAS,83,Distribution of Genes Classified Based on Abundance,2020/8/27,BIG-CAS,84,Probability of Transcript Detection,2020/8/

50、27,BIG-CAS,85,Transcriptome Statistics (YEAST),Total Species: 5460 transcripts Total Number: 15,000 polyA-RNAs per cell Average level: 2.8 copies/cellMedian level: 0.79 copies/cell 80% of the transcriptome at 0.1 - 2 mRNA copies/cell,2020/8/27,BIG-CAS,86,House-keeping Genes (HKG): Universal HKG (900

51、0) Tissue-specific HKG (500 x20) Cell-specific HKG (10-50 x200) Condition-related Genes (CRG): Development Differentiation Apoptosis Cell cycle Environment-regulated,Defining “House-keeping Genes”,2020/8/27,BIG-CAS,87,Defining the Transcriptome: Primary,Occurrence-definition Universal : shared by al

52、l tissues and cells Tissue-specific: shared by only limited number of tissues (such as nerve tissues, muscles, and epithelia) Cell-specific: unique to a cell type Near Universal: shared by most tissues but not all Rationally shared: genes that are shared between unrelated tissues or cell types based

53、 on function,2020/8/27,BIG-CAS,88,Expression-definition: Variability Expression-variable (majority; a gene varies in expression among tissues) Expression-constant (minority; a gene is expressed constantly in all cells) Expression-definition: Magnitude (1000s) Highly-expressed (1000s) Moderately-expr

54、essed (10s and 100s) Lowly-expressed (10 copies),Defining the Transcriptome: Primary,2020/8/27,BIG-CAS,89,Defining the Transcriptome: Primary,Function-definition: Gene structure Primary: Gene structure and composition Size: Large (500Kb) vs small; median size GC content: GC-rich vs GC-poor, CpG-cont

55、aining vs non-CpG-containing Pyrine-contant variations Secondary: Gene structure and organization CpG islands: high, moderate and low MI-containing Cluster-associated Secondary: Gene variation Mutation gradient-affected Gradient-unaffected Ka/Ks variations Secondary: Protein classes Olfactory recept

56、ors Transcription factor Kinases and phosphotases,2020/8/27,BIG-CAS,90,Continuing,Function-definition: Cellular Structure Mitochondrion-associated Chloroplast-associated Chromatin-associated Lamina-associated Nucleolus-associated Function-definition: Cellular Processes Circadian-regulated genes Cell

57、 cycle-regulated genes Metabolism-related genes RNA decay, Protein degradation, etc. Function-definition: Molecular Mechanisms Translation machinery Splicing machinery Transcription machinery Nuclear exporting machinery,2020/8/27,BIG-CAS,91,Defining the Transcriptome: Secondary,Condition-definition

58、Apoptosis Development Differentiation Phenotypic plasticity Environment-regulated Stem cell specific regulatory circuitry Cancer related,2020/8/27,BIG-CAS,92,Microarray-based HK Gene Lists,Warrington et al. assayed gene expression pattern of 11 tissues (8 tissues according to our classification) usi

59、ng Affymetrix HuGeneFL chip and cataloged 533 HK genes.,Hsiao et al. used the same microarray platform and obtained 451 HK genes by assaying 19 tissues. (18 tissues according to our classification),Eisenberg et al. used a dataset from Gene Expression Atlas I produced by more advanced Affymetrix U95A microarray platform and identified a set of 575 HK genes ubiquitously expressed in 47 tissues (about 30