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.

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

6. 20 June 20166

7. Susceptible
Resistant
Network Dynamics
7

8. Resistant Susceptible
24 Most Offending
Network Dynamics
8

9. 20 June 20169

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

11. 20 June 201611

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

18. 20 June 201618

19. 20 June 201619

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

23. RNA-Seq Databases
20 June 201623

24. 20 June 201624

25. 20 June 201625

26. 20 June 201626

27. Converting of sra format to fastq file
20 June 201627

28. 20 June 201628

29. 20 June 201629

30. 20 June 201630

31. 20 June 201631

32. 20 June 201632

33. 20 June 201633

34. Reference Genomes
20 June 201634

35. 20 June 201635

36. 20 June 201636

37. 20 June 201637

38. 20 June 201638

39. 20 June 201639

40. 20 June 201640

41. 20 June 201641

42. 20 June 201642

43. 20 June 201643

44. 20 June 201644

45. 20 June 201645

46. 20 June 201646

47. 20 June 201647

48. 20 June 201648

49. 20 June 201649

50. 20 June 201650

51. 20 June 201651

52. 20 June 201652

53. Special Thanks to:

54. Welcome to your questions….