HJAYHuman (Research) Junction Array

EURASNET, CambridgeSept. 14th, 2007

Tyson A. Clark

Typical Junction Array Design

Junction Arrays vs. Exon Arrays

Junction Exon

Pros

Cons

• Direct Measure of skipping events

• Reciprocal Analysis

• Multiple independent measurements of a single event

• Information on how exons are joined

• Ability to monitor small exons

• Increased genome coverage

• “Discovery” of Alt. Splicing

• Probe selection flexibility

• Comprehensive / Unbiased Design

• Observed events only (no discovery)

• Requires lots of probes

• Limited flexibility

• Half-Hyb

• Difficult to predict joining events without empirical evidence

• Cannot distinguish some isoforms

• Fewer probes per splicing event

• No joining information

Why use Exon-Exon Junction probes?

• Alt. spliced Exons (A & B) Alt. spliced Exons (A & B) present in 50% of transcriptspresent in 50% of transcripts

Why use Exon-Exon Junction probes?

• Alt. spliced Exons (A & B) Alt. spliced Exons (A & B) present in 50% of transcriptspresent in 50% of transcripts

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Why use Exon-Exon Junction probes?

• Alt. spliced Exons (A & B) Alt. spliced Exons (A & B) present in 50% of transcriptspresent in 50% of transcripts

- or - - or -

11

22

Why use Exon-Exon Junction probes?

• Alt. spliced Exons (A & B) Alt. spliced Exons (A & B) present in 50% of transcriptspresent in 50% of transcripts

- or - - or -

11

22

Cannot distinguishCannot distinguish

between situationbetween situation

1 & 21 & 2(using only exon representation)(using only exon representation)

?? ??

Advantages of Junction Probes

• Information on how exons are joined together• Exon skipping events are measured directly

– rather than just a decrease in exon signal– allows for reciprocal change analyses

• Exon-Exon junctions are non-genomic sequence– not present on a genome tiling array

• Ability to monitor small exons and distinguish alternative splice sites that are very close

HJAYDesign Information

New Research Junction Array DesignGenome-wide “Observed” Junctions

• Using content from ExonWalk (C. Sugnet), Ensembl, and RefSeq, we have designed an array that will include:– Exon Probes 8 – 12 PM probes per exon

– Exon – Exon Junction Probes 8 probes per junction (-4 to +4)

– >30,000 Human & Mouse Genes

– Human and Mouse designs will be manufactured onto separate chips

Design Input

• Human Input files (10,063,211 input exons)– (NCBI 36, March 2006 Genome Assembly)

• RefSeq (hNCBI36)• Ensembl (38)• ExonWalk (hNCBI36_exonwalkall)

• Mouse Input files (3,963,343 input exons)– (MM7, August 2005 Genome Assembly)

• RefSeq (mm7)• Ensembl (38)• ExonWalk (mm7_exonwalkall)

Exon Walk (developed by Chuck Sugnet)

• The ExonWalk program merges cDNA evidence together to predict full length isoforms, including alternative transcripts.

• ESTs Filtered– Present in cDNA libraries of another organism (i.e. also present in

mouse)– Or have three separate cDNA GenBank entries supporting it.

ExonWalk Transcripts – TCF7L2 as an example

Junction Design Strategy(Note reverse strand and non-overlapping transcripts are

separated into unique Transcript Clusters)

Genome Assembly

Genome Assembly

5' edge

3' edge

Observed Junctions

Input Transcripts (redundant)

Transcript Cluster (Non-redundant transcripts)

Exons and Exon Clusters (Non-redundant)

PSRs Non-redundant)

EX_C

EX_A

EX_B

541 2 3 6

New Junction Array – Design Example

Human Design Run #2 (1 week 1 day 41 minutes and 55 seconds elapsed)

• Transcript Clusters 35,123• with junctions 24,753

• Transcripts 335,663

• Junctions (Obs) 260,488• Exons 360,569• Exon Clusters 249,240• PSRs 315,137

Human DesignTranscript Cluster Content

35,123 "Genes"

Ensembl and Refseq and ExonWalk

1667348%Ensembl only

1106932%

Refseq only990%

ExonWalk only442813%

Ensembl and ExonWalk

14994%

Refseq and ExonWalk

1340%

Ensembl and Refseq12213%

Mouse Design (3 days 17 hrs 34 minutes and 1 seconds elapsed)

• Transcript Clusters 30,833• with junctions 25,431

• Transcripts 145,993

• Junctions (Obs) 237,871• Exons 319,769• Exon Clusters 239,114• PSRs 282,186

Mouse DesignTranscript Cluster Content

30,883 "Genes"

Ensembl and Refseq and ExonWalk

15,37650%

Refseq only5182%

Ensembl only680222%

ExonWalk only442813%

Ensembl and ExonWalk

22577%

Refseq and ExonWalk

5302% Ensembl and

Refseq16705%

One 49 Format 5 Micron Mask SetSplit Between Designs(6,553,600 features)

Note these are estimates pre-probe selection.

HJAY (Human Junction ArraY)Probes Per Oligos

Observed Junctions 260,488 8 2,083,904PSRs (Exon Probes) 315,137 8 2,521,096Tiling Targets 33,086 60 1,985,160Controls 75,000

Total 6,665,160

MJAY (Mouse Junction ArraY)Probes Per Oligos

Observed Junctions 237,871 8 1,902,968PSRs (Exon Probes) 282,186 10 2,821,860Tiling Targets 26,949 60 1,616,940Controls 75,000

Total 6,416,768

Tiling of adjacent intronic sequence

Tiling Targets1 50

100

150

200

230

G C T G A C T G C C A T A C C C C A C A C C T C C C T T C C C T A T A T G T G C C C A C A G C T T T T T A C C T C A C A G C T G T G C C C C T G T G G G G T G G C T G G A T T G C T A G G G C C A T T T C T C C C C A G G A G C C C C T G T G C T G G A T A A G T A G G G T C T T C C C T C A A G C T A A T A A A T A C T G C C C C C C A C C C C G G C C C T A C C A C A A T T T T A C C T A A A C C A C A C A T A A G T G C C T G C A A A G G G T T A A C C C C A C T C A ->

T A C C C C A C A C C T C C C T T C C C T A T A T T T T T A C C T C A C A G C T G T G C C C C T G T G G T G G C T G G A T T G C T A G G G C C A T T T C C T G T G C T G G A T A A G T A G G G T C T T C C C G G C C C T A C C A C A A T T T T A C C T A A A A G T G C C T G C A A A G G G T T A A C C C C A

C T T C C C T A T A T G T G C C C A C A G C T T T T G G C T G G A T T G C T A G G G C C A T T T C T G A T A A G T A G G G T C T T C C C T C A A G C T T A C C T A A A C C A C A C A T A A G T G C C T G T A A C C C C A C T C A ->C C C T C A A G C T A A T A A A T A C T G C C C C

231

250

300

301

G G G A T G G C A T A T G G T A G A A A A C A G G G T C T G A G G C T G G G C C C T C T C C C T T C T T G C C T T T C C T C A T G C A T A G

G G G A T G G C A T A T G T G G T A G A A A A C A G G G T C T G A G G C T G

T A T G G T A G A A A A C A G G G T C T G A G G C C C T T C T T G C C T T T C C T C A T G C A T A G

Exon

Tiling Probes Flanking the Exon (Only showing upstream and same strand) (ACE01009 Adam15)

Human Mousealt 3' splice sites 1,555 1,408alt 5' splice sites 904 807Cassette Exons 15,367 9,603Retained Intron 1,260 1,131

Conserved Exons 7,000 7,000Constitutive Exons 7,000 7,000

Total 33,086 26,949

Flanking Tiling Probes (15 per side, both strands)

Controls

Bg GC (Research) 20,595AFFX controls. 4,084

GC Bins (Antigenomic) 16,94320000_st 4,398cross_at 9

default_at 2,008default_st 9,098

norm_exon_pm_st 4,518norm_intron_pm_st 10,990

text_at 242text_st 493

Total Controls 73,378

Constitutive PSRs (Human, 116,284 constitutive, 36.9%)

Genome Assembly

Constitutive PSRs

EX_C

EX_A

EX_B

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PSRs (Probe Selection Regions)

Transcript Cluster

Exons / Exon Clusters1 2 3

Data Analysis

Analysis Approach

• Used the Splicing Index Algorithm treating each probeset (Exon or Junction) as independent– P-Value cutoff <0.001

– Magnitude of Change > |0.5| (log2 ratio ~1.4)

– 1570 Probesets in total passed those cutoffs

• Looked for Splicing Events that had more than 1 significant probeset– 252 genes with multiple probesets on the list– ~75% looked like real AS event

4 Probesets Monitor a Simple Cassette Exon

Some Examples

Highly Conserved 12bp Exon

CLASP1Event #1

CLASP1Event #2

Excel Sheet with Combined Results

221 Exons Total MED or higher confidence

• By Confidence Level– 26 HIGHEST

• All probesets from that splicing event made the list

– 89 HIGH• Reciprocal Junctions from the splicing event made the list

– 53 MED HIGH• Multiple Junctions from the splicing event made the list

– 53 MEDIUM

• By Splicing Event– 179 Cassette Exons

• 19 Mutually Exclusive Cassettes• 27 Multiple (consecutive) Cassettes

– 32 Alternative 3’ Terminal Exons– 5 Alt. 5’ss– 4 Alt. 3’ss

Histogram of Cassette Exon Size

16 Exons < 25 bp