Why Transcriptome? Why RNA-Seq? ENCODE answers….
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The document outlines a workshop on next-generation sequencing (RNA-seq), emphasizing its role in transcriptome analysis and the principles underlying systems biology and bioinformatics. It highlights the significance of the Encyclopedia of DNA Elements (ENCODE) project, which reshapes the understanding of gene function and regulation. RNA-seq is presented as a more comprehensive method compared to traditional microarrays, allowing for a detailed examination of transcript levels and variations.
Why Transcriptome? Why RNA-Seq? ENCODE answers….
- 2. Workshop on : “Next Generation Sequencing: The Basic Principles and Data Analysis of RNA-Seq” Why Transcriptome? Why RNA-Seq? ENCODE answers…. Mohammad Hossein Banabazi Scientific Board Department of Biotechnology Animal Science Research Institute of IRAN (ASRI) 15-17 April 2014
- 3. Concepts Systems Biology Bioinformatics Omics Data 20 June 20163
- 4. Systems Biology is about integrating data from different sources to provide a more comprehensive answer to a given biological question 20 June 20164
- 5. -ome Field of study (-omics) Collection of Exome Exomics Exons in a genome Genome Genomics (Classical genetics) Genes (DNA sequences/Chromosomes) Interactome Interactomics All interactions Metabolome Metabolomics Metabolites Metagenome Metagenomics Genetic material found in an environmental sample Phenome Phenomics Phenotypes Proteome Proteomics Proteins Transcriptome Transcriptomics mRNA transcripts Omics Data 20 June 20165
- 8. Resistant Susceptible 24 Most Offending Network Dynamics 8
- 10. The Encyclopedia of DNA Elements (ENCODE) is a public research project launched by the US National Human Genome Research Institute(NHGRI) in September 2003. Intended as a follow-up to the Human Genome Project (Genomic Research), the ENCODE project aims to identify all functional elements in the human genome. The project involves a worldwide consortium of research groups, and data generated from this project can be accessed through public databases. 20 June 201610
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- 12. Why Transcriptome? “ENCODE has played an important role in changing our concept of the gene,” A decade long project, the Encyclopedia of DNA Elements (ENCODE), has found that 80% of the human genome serves some purpose, biochemically speaking. Pennisi, E. (2012) ENCODE Project Writes Eulogy for Junk DNA, Science, 337, 1159-1161. 20 June 201612
- 13. Why Transcriptome? Pennisi, E. (2012) ENCODE Project Writes Eulogy for Junk DNA, Science, 337, 1159-1161. The ENCODE effort has revealed that a gene’s regulation is far more complex than previously thought, being influenced by multiple stretches of regulatory DNA located both near and far from the gene itself and by strands of RNA not translated into proteins, so-called noncoding RNA. 20 June 201613
- 14. Why Transcriptome? ENCODE has played an important role in changing our concept of the gene. As a result of ENCODE, the fundamental unit of the genome and the basic unit of heredity should be the transcript—the piece of RNA decoded from DNA—and not the gene. Pennisi, E. (2012) ENCODE Project Writes Eulogy for Junk DNA, Science, 337, 1159-1161. 20 June 201614
- 15. Nature Methods | VOL.5 NO.7 | JULY 2008 What’s RNA-Seq? a digital measure of the presence and prevalence of transcripts, in contrast to the analog-style signals obtained from fluorescent dye–based microarrays 20 June 201615
- 16. ???????????? ???????????? ???????????????????? ??????????? ??????????? ?????? ?????? ?????? ?????? ?????? ?????? ?????? ???? ????? AACGTT CTAACG TTAGCA ACCGAC ATGGCA TTGTCA CGCATG GTCACT Extract all mRNA Prepare a library of cDNA fragments Sequence fragments What’s RNA-Seq? RNAseq refers to the method of using Next-Generation Sequencing (NGS) technology to measure RNA levels. 20 June 201616
- 17. Align Sequences to Genome Gene A 17 Gene B Gene C GeneD TTGTCA CGCATG GTCACT TTAGCA ACCGAC ATGGCA AACGTT CTAACG Gene ID Sample1 A 3 B 3 C 0 D 2For a given gene, the number of reads aligned to the gene measures its expression level. 20 June 201617
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- 20. Some Advantages of RNAseq over Microarrays Microarrays measure only genes corresponding to predetermined probes on a microarray while RNAseq measures any transcripts in a sample. With RNAseq, there is no need to identify probes prior to measurement or to build a microarray. RNAseq provides count data which may be closer, at least in principle, to the amount of mRNA produced by a gene than the fluorescence measures produced with microarray technology. 20 June 201620
- 21. Some Advantages of RNAseq over Microarrays RNAseq provides information about transcript sequence in addition to information about transcript abundance. With RNAseq, it is possible to separately measure the expression of different transcripts that would be difficult to separately measure with microarray technology due to cross hybridization. Sequence information also permits the identification of alternative splicing, allele specific expression, single nucleotide polymorphisms (SNPs), and other forms of sequence variation. 20 June 201621
- 22. Further Reading: Flintoft, L. (2008) Transcriptomics: Digging deep with RNA-Seq, Nat Rev Genet, 9, 568-568. Haas, B.J. and Zody, M.C. (2010) Advancing RNA-seq analysis, Nature Biotech., 28, 421-423. Levin, J., et al. (2010) Development and evaluation of RNA-seq methods, Genome Biology, 11, P26. Marguerat, S. and Bahler, J. (2010) RNA-seq: from technology to biology, Cell. Mol. Life Sci., 67, 569- 579. Mortazavi, A., et al. (2008) Mapping and quantifying mammalian transcriptomes by RNA-Seq, Nat Methods, 5, 621-628. Wang, Z., Gerstein, M. and Snyder, M. (2009) RNA-seq: a revolutionary tool for transcriptomics, Nature Rev. Genet., 10, 57-63. Wilhelm, B.T., et al. (2010) Defining transcribed regions using RNA-seq, Nat Protoc, 5, 255-266. 20 June 201622
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- 54. Welcome to your questions….