RNA-SeqSebastian Groß

27.05.2015

Sebastian Groß 2

Why transcriptomics?

RNA-Seq: a revolutionary tool for transcriptomics

RNA-Seq benefits and comparison with other approaches

Challenges & opportunities

Direct RNA sequencing

Overview

27.05.2015

Sebastian 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?

27.05.2015

Complete set of transcripts and their quantitiy in one cell, or a population of

cells, for a specific devolpmental stage or physiological condition

Sebastian Groß 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

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Sebastian Groß 5

RNA isolation

RNA-Seq technology

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Sebastian Groß 6

RNA isolation

RNA preparation & (cDNA) library construction

RNA-Seq technology

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Sebastian Groß 7

RNA-Seq technology

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Sebastian 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

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Sebastian Groß 9

RNA-Seq technology

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Sebastian Groß 10

SOLiD

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Sequencing by oligonucleotide ligation and detection

developed by LifeTechnologies (Applied Biosystems)

Sebastian Groß 11

SOLiD

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Sebastian Groß 12

SOLiD

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Sebastian Groß 13

SOLiD

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Sebastian Groß 14

RNA-Seq benefits

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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

Sebastian Groß 15

RNA-Seq benefits

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RNA isoforms (alternative splicing)

Antisense transcription

Fusion genes

Sebastian Groß 16

Challenges & opportunities

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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

Sebastian Groß 17

Direct RNA sequencing

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Direct single molecule RNA sequencing without prior cDNA conversion

Detect femptomole (10-15) quantities of RNA

Sequencing by synthesis

Sebastian Groß 18

Direct RNA sequencing

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Polyadenylation by poly (A) polymerase I (PAPI) from E. coli

Blocking by 3‘ deoxyATP

Poly (A) tail ~ 150 nucleotides

Sebastian Groß 19

Direct RNA sequencing

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SourcesZ 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)

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