using isoform-sensitive microarrays to study different modes of alternative splicing

Using Isoform-Sensitive Using Isoform-Sensitive Microarrays to Study Different Microarrays to Study Different Modes of Alternative SplicingModes of Alternative Splicing

Christina ZhengChristina ZhengAres LabAres Lab

RNA Club RNA Club September 14, 2006September 14, 2006

OutlineOutline

Isoform-sensitive microarrays (splicing arrays)Isoform-sensitive microarrays (splicing arrays)– introductionintroduction– challengeschallenges

Probe cross-hybridizationProbe cross-hybridization– mapping of probes onto the genomemapping of probes onto the genome– excluding potential cross-hybridizing probesexcluding potential cross-hybridizing probes

Analysis of different modes of alternative Analysis of different modes of alternative splicingsplicing– annotation of different modesannotation of different modes– using splicing arrays to study different modesusing splicing arrays to study different modes

Isoform Ratio (IR)Isoform Ratio (IR)Isoform Expression (IE)Isoform Expression (IE)

Future directionsFuture directions

Splicing ArraysSplicing ArraysUsed to assay and identify splicing changes Used to assay and identify splicing changes associated with different biological conditionsassociated with different biological conditions– muscle specific alternative splicingmuscle specific alternative splicing– alternative splicing associated with nonsense mediated decayalternative splicing associated with nonsense mediated decay

The first splicing array was made in yeastThe first splicing array was made in yeast– Clark et al. Clark et al. Science Science 20022002

Mammalian splicing arraysMammalian splicing arrays– Johnson et. al. Science 2003– Pan et. al. Mol. Cell 2004– Li et. al. Li et. al. Cancer ResearchCancer Research 2006 2006– Le et. al. Le et. al. Nucleic Acids ResearchNucleic Acids Research 2004 2004– Sugnet et. al. Sugnet et. al. PLoSPLoS 2006 2006

Affymetrix Mouse Splicing Affymetrix Mouse Splicing ArrayArray

• 5 X106 25mer probes• probes are grouped intro probesets (6-10 probes)

• gene probesets - 8 – 10 probes placed in common regions• exon probesets• exon-exon junction probesets – 6 probesets across 30 nucleotides

• 15,000+ genes

Sugnet et al. PLoS Comput. Bio. 2006

inflexible probe selection• greater chance of cross-hyb

Splicing Arrays – AS eventsSplicing Arrays – AS events

All exon-exon junctions of human mRNA RefSeq

– Johnson et. al. Science 2003

Focused on simple cassette exon events – Pan et. al. Mol. Cell 2004

Focused on simple AS events with two isoforms

– Le et. al. Le et. al. Nucleic Acids ResearchNucleic Acids Research 2004 2004– Ule et al. Ule et al. Nature GeneticsNature Genetics 2005 2005– Sugnet et. al. Sugnet et. al. PLoSPLoS 2006 2006– Li et. al. Li et. al. Cancer ResearchCancer Research 2006 2006

Skip to include ratioSkip to include ratio– one measurement for each eventone measurement for each event– not applicable to more complicated modes of ASnot applicable to more complicated modes of AS

Difficulties with Splicing ArraysDifficulties with Splicing Arrays

Greater potential of probe cross-hybridizationGreater potential of probe cross-hybridization– inflexibility in probe selection due to location of eventsinflexibility in probe selection due to location of events

exon probes – restricted to the alternative exonexon probes – restricted to the alternative exon

exon-exon junction probes – restricted to exon-exon junctionexon-exon junction probes – restricted to exon-exon junction

Alternative splicing (AS) eventsAlternative splicing (AS) events– identifying/annotating themidentifying/annotating them– analyzing different modes of ASanalyzing different modes of AS

more complex with a greater number of isoformsmore complex with a greater number of isoforms

OutlineOutline






Probe RemappingProbe RemappingTools used to remap onto the May 2004 mouse Tools used to remap onto the May 2004 mouse assemblyassembly– GMAPGMAP Wu et al. Wu et al. BioinformaticsBioinformatics 2005 2005

– BLATBLAT – home-made junction databasehome-made junction database

used GMAP to align all mRNA and EST from unigeneused GMAP to align all mRNA and EST from unigenemade a database of sequences and genomic coordinates of all exon-exon made a database of sequences and genomic coordinates of all exon-exon junctions junctions

Remapped probesRemapped probes– uniquely mapped 25mer: 413502uniquely mapped 25mer: 413502– multiple hits: 25103 (cross-hyb to other genes)multiple hits: 25103 (cross-hyb to other genes)– not mapped 25mer: 62667not mapped 25mer: 62667

missed exon-exon junctionmissed exon-exon junctionSNPsSNPschanged from old mouse assembly to newchanged from old mouse assembly to new

Remapping ProbesRemapping Probes

Potential Cross-hybridizationPotential Cross-hybridization

Potential cross-hybridizationPotential cross-hybridization– BLAST ~400,000 uniquely mapped probesBLAST ~400,000 uniquely mapped probes

Cutoff for the level of similarity to other genesCutoff for the level of similarity to other genes– how do different levels of similarity affect probe intensity?how do different levels of similarity affect probe intensity?– took probes which only hit 2 genestook probes which only hit 2 genes

hit 25nt to one genehit 25nt to one genehit at different level to another (24nt, 23nt, 22nt ….)hit at different level to another (24nt, 23nt, 22nt ….)

– choose a cutoff based on the how the probe behavior in each classchoose a cutoff based on the how the probe behavior in each class

Probe AnalysisProbe Analysis

OutlineOutline






Analysis of Affy Splicing ArrayAnalysis of Affy Splicing Array

Previous workPrevious work– Ule et al. Ule et al. Nature GeneticsNature Genetics 2005 2005– Sugnet et al. Sugnet et al. PLoSPLoS 2006 2006

Focused on simple cassette exon events and Focused on simple cassette exon events and or simple two isoform eventsor simple two isoform events

Using a variation of skip to include ratioUsing a variation of skip to include ratio

Array was designed with more complicated Array was designed with more complicated eventsevents

Splicing Event Probe GroupingsSplicing Event Probe GroupingsAnnotated AS eventsAnnotated AS events– exonwalk exonwalk

identifies and annotates events, no matter how complicated the eventidentifies and annotates events, no matter how complicated the event

Mapped the probes onto annotated eventsMapped the probes onto annotated events

3418 AS events:3418 AS events:– 1 isoform: 2002 1 isoform: 2002 – 2 isoforms: 8922 isoforms: 892– 3 isoforms: 1823 isoforms: 182– 4 isoforms: 954 isoforms: 95– 5 isoforms: 445 isoforms: 44– 6 or more isoforms: 2036 or more isoforms: 203

Isoform RatioIsoform Ratio

Isoform 1 Isoform 2 Isoform 3

Isoform Ratio (IR) =isoform i isoform isoform = isoform1+isoform2+isoform3

isoform1 isoform

isoform2 isoform

isoform3 isoform


Significance Analysis of Microarrays (SAM)Significance Analysis of Microarrays (SAM)– identify statistically significant IRsidentify statistically significant IRs– based on a modified t test - ‘relative difference’based on a modified t test - ‘relative difference’

, , s = standard deviation; ss = standard deviation; s0 0 = small positive constant = small positive constant

q value - min false discovery rate (FDR) q value - min false discovery rate (FDR) Storey Storey J. Roy. Stat. Soc. Ser. BJ. Roy. Stat. Soc. Ser. B 2002 2002

– (FDR)(FDR)

– the minimum FDR incurred for calling a specific isoform significantthe minimum FDR incurred for calling a specific isoform significant– analogous to p-value for false positive rateanalogous to p-value for false positive rate– can use a q-value as a specific cutoff much like a p-valuecan use a q-value as a specific cutoff much like a p-value

xt - xc

s+s0

# of false positives

# of significant isoforms


Identifying muscle specific AS eventsIdentifying muscle specific AS events– C2C12 myoblast differentiation systemC2C12 myoblast differentiation system

Run samples on Affymetrix mouse splicing arrayRun samples on Affymetrix mouse splicing array

C2C12stem cells

differentiatestem cells

myo-tubule formation

isolate control RNA isolate test RNA

Analysis PipelineAnalysis PipelineBackground correction, normalization, and Background correction, normalization, and probe summarization probe summarization – RMA RMA (Irizarry et al. (Irizarry et al. Biostatistics Biostatistics 2003)2003)

Grouping probesets into splicing eventsGrouping probesets into splicing events– mapping probesets onto annotated AS eventsmapping probesets onto annotated AS events– calculating IRcalculating IR

Grouping probesets into genesGrouping probesets into genes– average of all probesets within a geneaverage of all probesets within a gene

Use SAM Use SAM (Tusher et al. (Tusher et al. PNAS PNAS 2001)2001) to test significance to test significance differences between test and controldifferences between test and control– q-value (min false discovery rate) q-value (min false discovery rate) Storey Storey J. Roy. Stat. Soc. Ser. BJ. Roy. Stat. Soc. Ser. B 2002 2002

Display results on dataviewerDisplay results on dataviewer

Splicing Array DataviewerSplicing Array Dataviewer

GeneViewerGeneViewer

Muscle Specific AS eventsMuscle Specific AS events

DnaJ (Hsp40) homolog

Coro6, actin binding protein

upregulated

upregulated

AAAAA

Multiple rounds of normal translationSTOP

Ribosome

include skip

AAAAA

STOP

EJC

Stop codon is in last exon

EJC EJC

Premature stop codon

(PTC)

>50nt

AAAAA

STOP

EJCEJC

AAAAA

NMDSTOP

Ribosome

EJC

Example: PTB

Isoform ExpressionIsoform Expression

Connection between AS and nonsense-mediated Connection between AS and nonsense-mediated decay (NMD)decay (NMD)

Block NMD and assay for changes in individual Block NMD and assay for changes in individual isoform changesisoform changes

Isoform ExpressionIsoform Expression

Isoform 1 Isoform 2 Isoform 3

Isoform Expression (IE) = log (isoform i) – log (gene) gene = probes in gene

log (isoform1) – log(gene) log (isoform2) – log(gene) log (isoform3) – log(gene)

Analysis PipelineAnalysis PipelineBackground correction, normalization, and Background correction, normalization, and probe summarization probe summarization – RMA RMA (Irizarry et al. (Irizarry et al. Biostatistics Biostatistics 2003)2003)

Grouping probesets into splicing eventsGrouping probesets into splicing events– mapping probesets onto predefined AS eventsmapping probesets onto predefined AS events– calculating IEcalculating IE

Grouping probesets into genesGrouping probesets into genes– average of all probesets within a geneaverage of all probesets within a gene

Use SAM Use SAM (Tusher et al. (Tusher et al. PNAS PNAS 2001)2001) to test the significance to test the significance between test and controlbetween test and control– q-value (min false discovery rate)q-value (min false discovery rate)

Display results on dataviewerDisplay results on dataviewer

AS associated with NMDAS associated with NMDSAT1SAT1 - spermidine/spermine N1-acetyl transferase 1 - spermidine/spermine N1-acetyl transferase 1 – down regulates polyamine levels in the celldown regulates polyamine levels in the cell– the inclusion of an alternative exon throws it out of frame NMDthe inclusion of an alternative exon throws it out of frame NMD– block NMD under conditions which SAT1 is neededblock NMD under conditions which SAT1 is needed

polyamine and polyamine analog (BENSPM) polyamine and polyamine analog (BENSPM)

expect inclusion of the exon the be repressedexpect inclusion of the exon the be repressed

– missed by previous analysis methods because this event is an example of missed by previous analysis methods because this event is an example of having probes for only one of the isoformshaving probes for only one of the isoforms

OutlineOutline






Probe cross-hybridizationProbe cross-hybridization– 18bp cross-hyb level18bp cross-hyb level– behavior of exon probes vs exon-exon junction probesbehavior of exon probes vs exon-exon junction probes

Different modes of ASDifferent modes of AS– better classification of the more complicated modesbetter classification of the more complicated modes

Future DirectionsFuture Directions

AcknowledgementsAcknowledgements

Ares LabManny Ares

John-Paul DonohueLeslie Grate

Roland NagelJulie Ni

Lily ShiueCharles Sugnet

Splicing ArraysSplicing Arrays

Clark et. al. Science 2002

40 nt probes40 nt probes

each intron-containing gene in yeasteach intron-containing gene in yeast

Splice Junction (SJ) Index = log - log (SJmut)

(SJwt)

(EXmut)

(EXwt)Normalize out gene expression

using isoform-sensitive microarrays to study different modes of alternative splicing

Documents

exonexon junctionalternative

mer probes probes

splicing changes

sugnet et

pan et

arraysjohnson et

common regions exon

probes gene