Case Study: RNASeq Sequencing

Chirag Nepal

23/03/2012

Outline

Introduction

Different kinds of experiment for transcriptome studies

RNA-Seq

ChiP-Seq

CAGE-Seq

Case studyHox-c cluster

Conclusion

High throughput sequencing (HTS)

HTS parallelize the sequencing process by producing millions of sequence at once.

Low cost, high throughput

Various sequencing machines

454 pyrosequencing

SOLiD sequencing

Illumina (Solexa) sequencing

• Applications

What is RNA-seq

RNA-seq is a revolutionary tool for transcriptomics studies with the use of high-throughput sequencing technologies to sequence cDNA, and gives information about a sample's RNA.

Major findings from RNA-Seq dataGene expression levelsAlternative splicing/isoformsIntron/exon boundariesDe-novo gene predictions

Library preparation for RNA-seq

Nature Methods - 5, 621 - 628 (2008

A typical pipeline for RNASeq analysis

Map reads to genome

Map splice reads

Construct gene model

Visualization

Differential expression

Multiple tools are available for each analysis

Use of multiple samples

Input sequence

• One experiment can produce tens of millions of such reads.

• Output from Illumina sequencing

Mapping of reads back to the genome

Nature Methods - 5, 621 - 628 (2008

• Challenges• Short (36bp- 76bp)• Multi mapping read

• Span exon boundary

UCSC genome browser

http://genome.ucsc.edu/ Step:1

Step:2

• Choose your species of interest

• Click custom tracks to add new tracks

Read coverage

Scalechr1:

2 kb208425000 208430000

RefSeq GenesNon-Rat RefSeq Genes

Ad-lib Rep-1

Vehicle Rep-1

Mus Malat1Homo MALAT1

Ad-lib Rep-1

918 _

1 _

Vehicle Rep-1

191 _

1 _

• Read counts gives an indication of gene expression level

Differential read count

Spliced reads

• Accounting spliced reads gives better intron/exon boundary

Merge the transcripts

Scalechr1:

TCONS_00002770TCONS_00002769TCONS_00002768

TCONS_00001998TCONS_00001997TCONS_00001999

TCONS_00002152TCONS_00002153

10 kb47180000 47190000 47200000

UCSC Known Genes Based on UniProt, RefSeq, and GenBank mRNAGene model

Gene model - Vehicle

Gene model - Adlib

• Differentially spliced• Alternative transcription start site PMID: 22383036

Have a look at the data in the browser.

RNASeq data between different replicates reveal a lot of dynamics

PMID: 22383036

Differential expression between samples

Gene of your interest

Questions ?