1. RNA-Seq Sebastian Groß 27.05.2015

2.  Why transcriptomics?  RNA-Seq: a revolutionary tool for transcriptomics  RNA-Seq benefits and comparison with other approaches  Challenges & opportunities  Direct RNA sequencing Overview 27.05.20152Sebastian Groß

3.  Interpretation of functional elements of the genome  Understanding development and disease  Determination of the transcriptional structure of genes:  Transcription start sites  5‘ and 3‘ ends  PTMs Why transcriptomics? 27.05.20153Sebastian Groß Complete set of transcripts and their quantitiy in one cell, or a population of cells, for a specific devolpmental stage or physiological condition

4.  Whole transcriptome shotgun sequencing  Uses next-generation sequencing methods:  Illumina IG (sequencing by synthesis)  Applied Biosystems SOLiD (sequencing by ligation)  Roche 454 Life Science (pyrosequencing) RNA-Seq 27.05.20154Sebastian Groß

5.  RNA isolation RNA-Seq technology 27.05.20155Sebastian Groß

6.  RNA isolation  RNA preparation & (cDNA) library construction RNA-Seq technology 27.05.20156Sebastian Groß

7. RNA-Seq technology 27.05.20157Sebastian Groß

8.  RNA isolation  RNA preparation & (cDNA) library construction  adaptor attachment to one or both ends  Next-generation sequencing  single-end sequencing  pair-end sequencing RNA-Seq technology 27.05.20158Sebastian Groß

9. RNA-Seq technology 27.05.20159Sebastian Groß

10. SOLiD 27.05.201510Sebastian Groß  Sequencing by oligonucleotide ligation and detection  developed by LifeTechnologies (Applied Biosystems)

11. SOLiD 27.05.201511Sebastian Groß

12. SOLiD 27.05.201512Sebastian Groß

13. SOLiD 27.05.201513Sebastian Groß

14. RNA-Seq benefits 27.05.201514Sebastian Groß  No reliance upon knowledge about genome sequence  attractive for non-model organisms  Very low background signal  No upper quantification limit  High level of reproducibility  Lower costs

15. RNA-Seq benefits 27.05.201515Sebastian Groß  RNA isoforms (alternative splicing)  Antisense transcription  Fusion genes

16. Challenges & opportunities 27.05.201516Sebastian Groß  Library construction & sequencing  Fragmentation of large RNA molecules  Reverse transcription  PCR artefacts  Bioinformatics  Reduction of error in image analysis  Removal of low quality-reads  Increase sequencing depth (e.g., longer reads)  Cost reduction

17. Direct RNA sequencing 27.05.201517Sebastian Groß  Direct single molecule RNA sequencing without prior cDNA conversion  Detect femptomole (10 -15 ) quantities of RNA  Sequencing by synthesis

18. Direct RNA sequencing 27.05.201518Sebastian Groß  Polyadenylation by poly (A) polymerase I (PAPI) from E. coli  Blocking by 3‘ deoxyATP  Poly (A) tail ~ 150 nucleotides

19. Direct RNA sequencing 27.05.201519Sebastian Groß

21. Sources Z Wang, M Gerstein, M Snyder (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics: Vol. 10, 57-63. F Ozsolak, M Milos (2011) RNA sequencing: advances, challanges and opportunities. Nature Reviews Genetics: Vol. 12, 87-98. A Mortazavi (2015) Better together: multiplexing samples to improve the preparation and reliability of gene expression studies. Nature Methods: Vol. 12, No. 4, 304-305. ML Metzker (2010) Sequencing technologies - the next generation. Nature Reviews Genetics: Vol. 11, 21-46. F Ozsolak, AR Platt, DR Jones, JG Reifenberger, LE Sass, P Mclnemey, JF Thompson, J Bowers, MJ Milos, PM Milos (2009) Direct RNA sequencing. Nature: Vol. 468, 814-818. P Chomczynski, Nicoletta Sacchi (1987) Single ‑ Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol- Chloroform Extraction. Analytical Biochemistry: 162, 156-159. HE Peckham, SF McLaughlin, JN Ni, MD Rhodes, JA Malek, KJ McKernan, AP Blanchard. SOLiD Sequencing and 2 ‑ Base Encoding. Applied Biosystems (Poster 2624). http://www3.appliedbiosystems.com/cms/groups/mcb_marketing/documents/generaldocuments/cms_057810.pdf (27.05.2015) http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture9/AMG5.1a.gif (19.05.15) http://image.slidesharecdn.com/htsmasterstalk ‑ 111109074414 ‑ phpapp02/95/highthroughput-sequencing-10- 728.jpg%3Fcb%3D1320824865 (20.05.15) http://ll1.workcast.net/10403/4934292334051602/images/10403_rna_img.jpg (22.05.15) http://www.howfelonscangetjobs.com/p/ask-me-question.html (27.05.15)