逢甲大學 生物資訊研究中心

 

Constructions and Applications of Constructions and Applications of Alternative Splicing DatabasesAlternative Splicing Databases

speaker: speaker:  許芳榮許芳榮

OutlineOutline

IntroductionIntroductionConstruction of alternative splicing Construction of alternative splicing

databasedatabaseSurvey of existing solutionsSurvey of existing solutionsApplications Applications

IntroductionIntroduction

RNA SplicingRNA Splicing

Alternative SplicingAlternative Splicing

DefinitionsDefinitionsSplicing the Splicing the same pre-mRNAsame pre-mRNA in in two or two or more waysmore ways to yield two or more to yield two or more different different mRNAsmRNAs that produce two or more that produce two or more different different protein productsprotein products

Types of alternative splicingTypes of alternative splicing

The Troponin T (muscle protein) pre-mRNAis alternatively spliced to give rise to64 different isoforms of the protein

Constitutively spliced exons (exons 1-3, 9-15, and 18)

Mutually exclusive exons (exons 16 and 17)

Alternatively spliced exons (exons 4-8)

Exons 4-8 are spliced in every possible waygiving rise to 32 different possibilities

Exons 16 and 17, which are mutually exclusive,double the possibilities; hence 64 isoforms

EExpressed xpressed SSequence equence TTags (ESTs)ags (ESTs)

What are the relationships What are the relationships of Genome, mRNA and ESTof Genome, mRNA and EST

s?s?

Genome

EST

EST

3’

Exon 1 Exon 2 Exon 3 Exon 4Intron 1 Intron 2 Intron 3

5’

AAA...

EST 1

EST 2

EST 4

EST 3

EST 5

EST 6

AAA...EST 7

Construction of alternative Construction of alternative splicing databasesplicing database

Gene Discovery

SNPAlternative

Splicing

dbEST

5 million ESTs

Genome Sequences

3 billion bp

alignment

Exons, Introns Database

Methods of Alternative Methods of Alternative Splicing Detection Splicing Detection

mRNA – EST alignment (or EST mRNA – EST alignment (or EST consensus)consensus)Without knowledge of genomic Without knowledge of genomic

sequencesequenceGenomic sequence to EST alignmentGenomic sequence to EST alignment

informativeinformative

How to cluster ESTs ? How to cluster ESTs ? UniGene clusterUniGene cluster

Consider the ESTs in the same UniGene clusConsider the ESTs in the same UniGene clusterter

Save time but not informativeSave time but not informativeGenome templateGenome templateGenomic sequence to EST alignmentGenomic sequence to EST alignment

informative but time consuming informative but time consuming

The Approaches of EST The Approaches of EST ClusteringClustering

Unigene like approachUnigene like approach1.1. Overlapped ESTs are grouped in a cluster as UnigOverlapped ESTs are grouped in a cluster as Unig

ene.ene.2.2. Generating a consensus sequence of each cluster.Generating a consensus sequence of each cluster.3.3. Aligning consensus sequences to genome sequeAligning consensus sequences to genome seque

nce. nce. Genome template Genome template

1.1. Cut Human Genome Sequence in 20k base pairs.Cut Human Genome Sequence in 20k base pairs.2.2. Screening in ESTs similarity by BLAST.Screening in ESTs similarity by BLAST.3.3. Detecting exons by sim4. Detecting exons by sim4.

Directly alignmentDirectly alignment

Unigene like approachUnigene like approach

1.1. Overlapped ESTs aOverlapped ESTs are grouped in a clure grouped in a cluster as Unigene.ster as Unigene.

2.2. Generating a consGenerating a consensus sequence oensus sequence of each cluster.f each cluster.

3.3. Aligning consensuAligning consensus sequences to ges sequences to genome sequence. nome sequence.

genomic seq

Candidates of gene location

STS

BLAST

gene

consensus sequence

Report exons

Genome templateGenome template

1.1. Cut Human GenoCut Human Genome Sequence into me Sequence into 20k base pairs.20k base pairs.

2.2. Screening in ESTs Screening in ESTs similarity by BLASsimilarity by BLAST.T.

3.3. Detecting exons bDetecting exons by sim4. y sim4.

genomic template

EST DB

WU-BLAST

ESTs with similarity

Sim4

exons

Directly alignmentDirectly alignment

Using UniGene Cluster is not InfUsing UniGene Cluster is not Informativeormative

Many ESTs in different UniGene clusteMany ESTs in different UniGene clusters are aligned to same genome area.rs are aligned to same genome area.

UniGene cluster ID UniGene cluster ID 101131,100437,100101131,100437,100738,101182 and 100143738,101182 and 100143 should be gro should be grouped together to detect alternative spuped together to detect alternative splicinglicing

Resource Description Approach Quantity Species Institute

ASAP

performed genome-wide detection of human

alternative splicing and Unigen Like6201 A.S sites inall genes Human UCLA

TAP

performed an EST-based gene structure

prediction in genomic sequences and alsocollected splicing information Genome template

669 A.S sites in365of the 1007multiexon genes Human WU

PALSdb

PALS db is a collection of PutativeAlternative Splicing information. Alternative

splicing sites were predicted by using the

longest mRNA sequence in each UniGenecluster as the reference sequence Unigen Like 9,952/19,936

Human,mouse Yang Ming

Compugen

ESTs and all mRNA sequences were alignedwith the human genomesequence using LEADS, Compugen’salternative splicing modeling platform. Unigen Like Human Compugen

SpliceNest

Web-based graphical tool to explore genestructure, including alternative splicing,based on a mapping of the EST consensusfrom GeneNest to complete human genome Unigen Like

26880 introns,32348 exons from5468 genes,

Human,mouse,arabidopsis,zebrafish

Max-Planck-Gesellschaft

STACKSTACK can provide putative tissue-specifictranscripts for each gene Unigen Like Human

Egenetic,SouthAfrica

Avatar: Avatar: aa vvalue alue aadded dded ttrraanstnstrriptomiptome databasee database

Align entire dbEST to genome using PCsAlign entire dbEST to genome using PCs

Homo sapiens 14,989 22,969 11,188 330 7,481

Mus musculus 7,479 13,075 4,850 127 3,493

Rattus norvegicus 531 900 401 4 373

Caenorhabditis elegans

162 28 263 5 174

Drosophila melanogaster

351 117 221 6 221

Arabidopsis thaliana 83 4 77 1 32

OrganismNumber. of alternative splicing events

5’ AS 3’ ASExon

skippingmutually exclusive

intron retention

ApplicationsApplications

Cross-species analysisCross-species analysisTissue specific analysisTissue specific analysisSNP and alternative splicing SNP and alternative splicing Quantity analysis Quantity analysis Splicing enhancer Splicing enhancer Gene prediction through dbESTGene prediction through dbESTSNP finding through dbESTSNP finding through dbEST

BoneMarrow , 1

Brain , 15

Eye , 1

Liver, 15

Lung , 5

LymphNode , 1

MammaryGland , 1

Placenta , 4

Stomach , 5

Testis, 1

WholeBlood, 2

BoneMarrow Brain Eye LiverLung LymphNode MammaryGland Placenta Stomach TestisWholeBlood

Tissue distributions of 51 tumor-specific alternative splicing sites

1,598 SNP dependent alternative 1,598 SNP dependent alternative splicingsplicing

Comparison of human and Comparison of human and micemice

Exon skipping Exon skipping

Conserved alternative splicing events (CES events)

F1

F2

F1

F2

F1

F2

F1

F2

If NCES.F1 > K and NCES.F2 == 0

Non-conserved alternative splicing events (NCES events)

Discovering the different Discovering the different constitutive splicing eventsconstitutive splicing events

94 91

ME12713588-1 ME12751459-1

HumanSNX3

MR12705131-1

EST support: 41

ME2231614-2

MouseSnx3

ME2238811-1 EST support: 90

+

EST frequency >=10

EST frequency >=1

PSMD13

Psmd13

F2

F1

GGTGAACCCTTTGTCCCTCGTGGAAATCATTCTTCATGTAGTTAGACAGATGACTG

GGTAAACCCTCTGTCCCTGGTAGAAATAATTCTCCATGTGGTTAGACAGATGACCG

MR178998-1 ME184041-1 ME184161-1

ME579264-1

ME582152-1 ME582275-1

184167,C,T,D,2,2,48,0,0.00452488687782805

184171,T,C,D,2,2,48,0,0.00452488687782805

Human exon

Mouse exon

F1

86CT

CT

TC

T C

48

2 2

0

Finding SNP from dbESTFinding SNP from dbEST

3’

Exon 1 Exon 2 Exon 3 Exon 4Intron 1 Intron 2 Intron 3

5’

AAA...

EST 1

EST 2

EST 4

EST 3

EST 5

EST 6

AAA...EST 7

EST to genome alignment with EST to genome alignment with profileprofile

3’

Exon 1 Exon 2 Exon 3 Exon 4Intron 1 Intron 2 Intron 3

5’

EST 4

EST 3

EST 5

EST 6

AAA...EST 7

TranslocationTranslocation

Finding gene from dbESTFinding gene from dbEST

3’

Exon 1 Exon 2 Exon 3 Exon 4Intron 1 Intron 2 Intron 3

5’

AAA...

EST 1

EST 2

EST 4

EST 3

EST 5

EST 6

AAA...EST 7

Transciptome GenomicsTransciptome GenomicsWhere Where What What Why Why How How

ConclusionConclusion