Paola Bonizzoni Graziano Pesole * Raffaella Rizzi

15-20 september WABI03

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A Method to Detect Gene Structure and Alternative

Splice Sites by Agreeing ESTs to a Genomic Sequence

Paola Bonizzoni Graziano Pesole* Raffaella Rizzi

DISCo, University of Milan-Bicocca, Italy*Department of Physiology and Biochemistry, University of

Milan, Italy

Supported by FIRB Bioinformatics: Genomics and Proteomics


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Outline Gene structure and alternative

splicing (AS) Problem definition and algorithm ASPic program Experimental results and

discussion


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Mechanism of Splicing3’

5’

5’

3’DNA

TRANSCRIPTION

5’

3’

exon 1 exon 2 exon 3pre-mRNA

SPLICING by spliceosome

exon 1 exon 2 exon 3 splicing productmRNA

EST Expressed Sequence Tag(cDNA)

exon 2exon 1 exon 3


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Modes of Alternative Splicing

1 2 3

Genomic sequence

1 2 3

ExonsIntrons

1 2 3

First splicing modeSecond splicing mode

1 3

Third splicing mode

2 3


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Modes of Alternative Splicing

1 2 32b

Competing 5’–3’

Exclusive exons: 1 31 2b


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Why AS is important? AS occurs in 59% of human genes

(Graveley, 2001) AS expands protein diversity

(generates from a single gene multiple transcripts)

AS is tissue-specific (Graveley, 2001)

AS is related to human diseases


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Motivations

predict alternative splicing forms analyze such a mechanism by a representation of splicing forms

Regulation of AS is still an open problem

NEED tools to


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What is available?Fast programs to produce a single EST alignment to a genomic sequence: Spidey (Wheelan et al., 2001)

Squall (Ogasawara & Morishita, 2002)

But to predict the exon-intron gene structure is acomplicate goal because of

sequencing errors in EST make difficult to locate splice sites by alignment duplications, repeated sequences may produce more than one possible EST

alignment


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Open Problems Formal definition of AS prediction problem

…

Combined analysis of ESTs alignments related to the same gene by agreeing ESTs to a common exon-intron gene structure

Optimization criteria


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Formal Definitions Def 1

Genomic sequence, G = I1 f1 I2 f2 I3 f3 … In fn In+1, where Ii (i=1, 2, …, n+1) are introns and fi (i=1, 2, …, n) are exons

Def 2 Exon factorization of G, GE = f1 f2 f3 … fn

Def 3 EST factorization of an EST S compatible with GE is

S=s1 s2 … sk s.t. there exists 1 i1 < i2 < … < ik n:

st = fit for t=2, 3, …, k-1 s1 is a suffix of fi1 and sk is a prefix of fik

st = suff (fit) or st = pref (fit)splice variant

Def 1 Genomic sequence, G = I1 f1 I2 f2 I3 f3 … In fn In+1, where Ii

(i=1, 2, …, n+1) are introns and fi (i=1, 2, …, n) are exons Def 2

Exon factorization of G, GE = f1 f2 f3 … fn Def 3

EST factorization of an EST S compatible with GE is S=s1 s2 … sk s.t. there exists 1 i1 < i2 < … < ik n:

edit (st, fit) error for t=2, 3, …, k-1 edit(s1, suff(fi1)) error and edit(sk, pref(fik)) error


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The ProblemInput

- A genomic sequence G- A set of EST sequences S = {S1, S2, …, Sn}

Output

An exon factorization GE of G (GE = f1, f2, …, fn) and aset of ESTs factorizations compatible with GE

Objective: minimize n


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ExampleGenomic sequence G

EST set S = {S1, S2, S3}

S2 A1A2 B D1

S3 A2 D1D2 C1C2

A2 A1A2 B D1 C1 D1D2 C1C2

C1S1 A2 D1

A2 D1 C1A2 D1 C1A1A2 B D1A1A2 B D1A2 D1D2 C1C2A2 D1D2 C1C2

7 exons

B D1D2 C1C2

4 exons

A1A2


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Results MEFC is MAX-SNP-hard (linear

reduction from NODE-COVER)

heuristic algorithm: Iterate process to factorize each ESTbacktracking to recompute previous EST factorsif not compatible to GE


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The algorithm

si1 si j-1 sijSi

e1 e2G

Iterative jth step: partial EST factorization of Si (compute factor sij)

em

if (Compatible(em, exon_list)) thenadd em to exon_list;

otherwise try to place sij elsewhere;

em

If not possible then backtrack;

si-1 1 si-1 j-1 si-1 j si-1 nSi-1

After placing all the factors sij for the set S,place the external factors;


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The algorithm (more details)

G

si1 si j-1Sisi j

Compute factor sij

Sij can be divided into n components ck (k=1,2,…,n)At least one of these components for k from 1 to (n-1)is error-free and can be placed on G

sijc1 c2 c3 c4 c5

The algorithm searches a perfect match of c1 on G

c1

Suppose that c1 has no perfect match on G

Then the algorithm searches a perfect match of c2 on G

c2c1c1

Suppose that c2 has a perfect match on G

c2

Then the entire factor sij can be placed on GFind the canonical ag pattern on the left

ag

Find the rightmost gt pattern such that the edit distance between sijy and the genomic substring from ag to gt is bounded

gt

si jy

exon


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ASPic (Alternative Splicing PredICtion)

Input- A minimum length of an exon- A maximum number of exons in the exon factorization of the genomic sequence- An error percentage- A genomic sequence- An ESTs set (or cluster)

Output- A text file for all ESTs alignments- An HTML file for the exon factorization of the genomic sequence


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ASPic data validation

ASAP (Lee et al., 2003)

Genomic sequences from ASAP database EST clusters of human chromosome 1 from UniGene database

ASPic INPUT:

Validation Database:


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Experimental Results

Genomic sequence(official gene name)

Introns detectedby ASAPASAP intronsdetected by ASPic

Novel introns detectedby ASPic

Genomic shift detected by ASPic


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Execution timesPENTIUM IV, 1600 MHZ, 256 MB, running Linux


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An example of data (gene HNRPR)

ASPic finds a novel intronfrom 2144 to 5333 confirmedby 18 EST sequencesPositions are from 0 for ASPic and from 1 for ASAP


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An example of data (gene HNRPR, intron 2144-5333)

EST ID

Left and right ends of thetwo exonsEST exonsGenomic exons


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WEB site


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WEB site


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WEB site


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Responsabili di progetto: Prof. Paola Bonizzoni Prof. Graziano Pesole

Responsabile disegno software: Raffaella Rizzi

Sito WEB:Gabriele RavanelliRappresentazione grafica: Francesco Perego

Anna RedondiAnalisi dati: Francesca RossinAltri contributi: Gianluca Dellavedova


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GRAZIE!

Documents

Paola Bonizzoni Graziano Pesole * Raffaella Rizzi