Transcriptomics : A general overview By Todd, Mark, and Tom

1. Transcriptomics:A general overviewBy Todd, Mark, and Tom

2. Intro • Transcriptomics => RNA in a cell • Either coding or non-coding (ncRNA). mRNA vs microRNA, siRNA Also non-functional RNA (pseudo-genes)

3. Transcriptomics focuses sets on:howwherewhenwhyAlso diagnosing :developmental stagestissue differentialsvirusesresponse to stimuli

4. Microarray • Used for Biological Assays • DNA Microarrays • MMChips • Protein Microarrays • Tissue Microarrays • Antibody Microarrays

5. DNA Microarray • Can be used to measure • Expression levels • SNPs • Genotyping • Comparative Genome Hybridization

6. Basic DNA Microarray Experiment http://en.wikipedia.org/

7. Labeling Lockhart and Winzeler 2000

8. Probes and Targets • Probes • Known sequence bonded to substrate • Target • Sample obtained to wash over chip • See what and how much is hybridized

9. Hybridization and Wash

10. Hybridization and Wash

11. Basic DNA Microarray Experiment http://en.wikipedia.org/

12. Results Lockhart and Winzeler 2000

13. Tiling Array • Genome array consisting of overlapping probes • Finer Resolution • Better at finding RNA in the cell • mRNA • Alternative splicing • Not Polyadenylated • miRNA

14. Tiling Arrays http://en.wikipedia.org/

15. Tiling Array http://en.wikipedia.org/

16. Microarray Wheelan et al. 2008

17. Gene expression profiling predicts clinical outcome of breast cancerLaura J. van 't Veer1,2, Hongyue Dai2,3, Marc J. van de Vijver1,2, Yudong D. He3, Augustinus A. M. Hart1, Mao Mao3, Hans L. Peterse1, Karin van der Kooy1, Matthew J. Marton3, Anke T. Witteveen1, George J. Schreiber3, Ron M. Kerkhoven1, Chris Roberts3, Peter S. Linsley3, René Bernards1 and Stephen H. Friend3 Divisions of Diagnostic Oncology, Radiotherapy and Molecular Carcinogenesis and Center for Biomedical Genetics, The Netherlands Cancer Institute, 121 Plesmanlaan, 1066 CX Amsterdam, The Netherlands Rosetta Inpharmatics, 12040 115th Avenue NE, Kirkland, Washington 98034, USA These authors contributed equally to this workNature, January 2002

18. • Use DNA microarray analysis and applied supervised classification to identify a gene expression signature predictive of metastases and BRCA1 carriers. • Authors predicted that the expression profile would outperform all currently used clinical parameters in predicting disease outcome. • Strategy to select patients who would benefit from adjuvant therapy (chemotherapy).

19. Metastases – spread of cancer from one area to another; characteristic of malignant tumor cells. Angiogenesis – process of growing new blood vessels from pre-existing vessels. A normal process in growth and development, however also a fundamental step in the transition of tumors from a dormant state to a malignant state. Estrogen Receptor alpha (ERα) – activated by sex hormone estrogen; DNA binding transcription factor which regulates gene expression; association with cancer known from immunohistochemical data (IHC).BRCA1 – Human gene, Breast Cancer 1; Mutations associated with significant increase in risk of breast cancer. • Belongs to a class of genes known as tumor suppressors (DNA damage repair, transcriptional regulation). • BRCA1 represses ERα-mediated transcription, with a reduction of BRCA1 activity results in elevated ERα-mediated transcription and enhanced cell proliferation.

20. 98 primary breast cancers: 34 from patients who developed metastases within 5 years44 from patients who continued to be disease-free after 5 years18 from patients with BRCA1 germline mutations 2 from BRCA2 carriers • Total RNA isolated from patients and used to derive complementary RNA (cRNA) • A reference cRNA pool was made by pooling equal amounts of cRNA from each cancer, for use in quantification of transcript abundance (fluorescence intensity in relation to reference pool). • Hybridizations carried out on micoarrays (synthesized by inkjet technology) containing ~ 25,000 human genes • ~ 5,000 genes found to be significantly regulated across the group of samples

21. Two distinct groups of tumours apparent on the basis of the set of ~5,000 significant genes. • In upper group only 34% of patients were from group developing metastases within 5 years. • In lower group 70% of patients had progressive disease. • Clustering detects two subgroups of cancer which differ in ER status and lymphocytic infiltration

22. 1) The correlation coefficient of the expression of ~ 5,000 significant genes was calculated, with 231 genes determined to be significantly associated with disease outcome.2) These 231 genes were ranked on basis of magnitude.3) Number of genes in ‘prognosis classifier’ optimized with the optimal number of marker genes reached at 70 genes. To identify tumours that could reliably represent either a good or poor prognosis a three-step supervised classification method was applied:

23. Prognosis signature with prognostic reporter genes identifying two types of disease outcome: • above dashed line good prognosis • below dashed line poor prognosis Predicted correctly the actual outcome of disease for 65 out of 78 patients (83%). To validate prognosis classifier additional set analyzed (Fig. 2C).

24. The functional annotation of genes provided insight into the underlying mechanisms leading to rapid metastases with the following genes significantly upregulated in the poor prognosis signiture: • genes involved in cell cycle • invasion and metastasis • angiogenesis • signal transduction

25. A third classification was performed to look at the expression patterns associated with ER-positive and ER-negative tumours.ER clustering has predictive power for prognosis although it does not reach the level of significance of the prognosis classifier.

26. Consensus conference developed guidelines for eligibility of adjuvant chemotherapy based on histological and clinical characteristics. Prognosis classifier selects as effectively high-risk patients who would benefit from therapy, but reduces number to receive unnecessary treatment.

27. • Results indicate that breast cancer prognosis can be derived from gene expression profile of primary tumor. Conclusions: Recogmendations: • ER signature - can be used to decide on hormonal therapy • BRCA1 - knowing status of can improve diagnosis of hereditary breast cancer. • Genes overexpressed in tumors with poor prognosis profile are targets for development of new cancer drugs

28. MicroRNA expression profiles classify human cancers Jun Lu1,4*, Gad Getz1*, Eric A. Miska2*†, Ezequiel Alvarez-Saavedra2, Justin Lamb1, David Peck1, Alejandro Sweet-Cordero3,4, Benjamin L. Ebert1,4, Raymond H. Mak1,4, Adolfo A. Ferrando4, James R. Downing5, Tyler Jacks2,3, H. Robert Horvitz2 & Todd R. Golub1,4,6 Nature, June 2005

29. Short size of microRNAs (miRNAs) and sequence similarity between miRNA family members has resulted in cross-hybridization of related miRNAs on glass-slide microarrays. Development of bead-based flow cytometric expression profiling of miRNAs.

31. miRNA profiles are informative with a general down regulation of miRNA in tumors compared with normal tissueExpression profiles of miRNA are also able to classify poorly differentiated tumors, highlighting the potential for miRNA profiling in cancer diagnosis

33. RNA-Seq Lockhart and Winzeler 2000 Wang et al. 2009

34. RNA-Seq • Whole Transcriptome Shotgun Sequencing • Sequencing cDNA • Using NexGen technology • Revolutionary Tool for Transcriptomics • More precise measurements • Ability to do large scale experiments with little starting material

35. RNA-Seq Experiment Wang et al. 2009

36. Mapping • Place reads onto a known genomic scaffold • Requires known genome and depends on accuracy of the reference http://en.wikipedia.org/

37. Mapping • Create unique scaffolds • Harder algorithms with such short reads

38. Comparisons Wang et al. 2009

39. Comparisons Wang et al. 2009

40. Biases Wang et al. 2009

41. Directionality Wang et al. 2009

42. Coverage Versus Depth Wang et al. 2009

43. New Insights • Mapping Genes and Exon Boundries • Single Base Resolution • Transcript Complexity • Exon Skipping • Novel Transcription • More accurate • No cross hybridization

44. Transcription Levels • Can measure Transcript levels more accurately • Confirmed with qPCR and RNA spike-in • Can compare measurements with different cellular states and environmental conditions • Without sophistication of normalization of data

45. What does mRNA tell you? Gene expression not the same as phenotypic expression

46. Why no line? Reasons? • Noise and bias of sample • Lag time of translation • Post-translational control • RNA/Protein half life • ?

47. Where is the genetics? • How do you study the transcriptome? • What are the patterns of expression telling you? • Differences between gene expression vs gene function (i. e. protein code vs concentration)?

48. 1) ‘guilt by association’ 2) Change environment, look for patterns; compare known phenotypic mutants (cancer) 3) Add ‘controlled’ knockout (specific locations/ times/ concentrations) 4)Evolution; diversity of expression across intra and inter speices 5) Add entire chromosome

49. Evolution model: neutral vs selection