RNA-Seq Sebastian Groß 27.05.2015.  Why transcriptomics?  RNA-Seq: a revolutionary tool for transcriptomics  RNA-Seq benefits and comparison with other.

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Presentation transcript:

RNA-Seq Sebastian Groß

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

 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? Sebastian Groß Complete set of transcripts and their quantitiy in one cell, or a population of cells, for a specific devolpmental stage or physiological condition

 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 Sebastian Groß

 RNA isolation RNA-Seq technology Sebastian Groß

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

RNA-Seq technology Sebastian Groß

 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 Sebastian Groß

RNA-Seq technology Sebastian Groß

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

SOLiD Sebastian Groß

SOLiD Sebastian Groß

SOLiD Sebastian Groß

RNA-Seq benefits Sebastian 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

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

Challenges & opportunities Sebastian 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

Direct RNA sequencing Sebastian Groß  Direct single molecule RNA sequencing without prior cDNA conversion  Detect femptomole ( ) quantities of RNA  Sequencing by synthesis

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

Direct RNA sequencing Sebastian Groß

Sources Z Wang, M Gerstein, M Snyder (2009) RNA-Seq: a revolutionary tool for transcriptomics. Nature Reviews Genetics: Vol. 10, F Ozsolak, M Milos (2011) RNA sequencing: advances, challanges and opportunities. Nature Reviews Genetics: Vol. 12, A Mortazavi (2015) Better together: multiplexing samples to improve the preparation and reliability of gene expression studies. Nature Methods: Vol. 12, No. 4, ML Metzker (2010) Sequencing technologies - the next generation. Nature Reviews Genetics: Vol. 11, 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, P Chomczynski, Nicoletta Sacchi (1987) Single ‑ Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol- Chloroform Extraction. Analytical Biochemistry: 162, HE Peckham, SF McLaughlin, JN Ni, MD Rhodes, JA Malek, KJ McKernan, AP Blanchard. SOLiD Sequencing and 2 ‑ Base Encoding. Applied Biosystems (Poster 2624). ( ) ( ) ‑ ‑ phpapp02/95/highthroughput-sequencing jpg%3Fcb%3D ( ) ( ) ( )