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HJAY Human (Research) Junction Array

HJAY Human (Research) Junction Array. EURASNET, Cambridge Sept. 14 th , 2007 Tyson A. Clark. Typical Junction Array Design. Junction Arrays vs. Exon Arrays. Exon. Junction. Direct Measure of skipping events Reciprocal Analysis Multiple independent measurements of a single event

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HJAY Human (Research) Junction Array

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  1. HJAYHuman (Research) Junction Array EURASNET, Cambridge Sept. 14th, 2007 Tyson A. Clark

  2. Typical Junction Array Design

  3. Junction Arrays vs. Exon Arrays Exon Junction • 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 Pros • 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 Cons

  4. Why use Exon-Exon Junction probes? • Alt. spliced Exons (A & B) present in 50% of transcripts

  5. Why use Exon-Exon Junction probes? • Alt. spliced Exons (A & B) present in 50% of transcripts 1

  6. Why use Exon-Exon Junction probes? • Alt. spliced Exons (A & B) present in 50% of transcripts 1 - or - 2

  7. Why use Exon-Exon Junction probes? • Alt. spliced Exons (A & B) present in 50% of transcripts 1 Cannot distinguish between situation 1 & 2 (using only exon representation) ? ? - or - 2

  8. 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

  9. HJAYDesign Information

  10. 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

  11. 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)

  12. 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.

  13. ExonWalk Transcripts – TCF7L2 as an example

  14. Junction Design Strategy(Note reverse strand and non-overlapping transcripts areseparated into unique Transcript Clusters)

  15. New Junction Array – Design Example

  16. 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

  17. 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

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

  19. Tiling of adjacent intronic sequence

  20. Tiling Targets

  21. Controls

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

  23. Data Analysis

  24. 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

  25. 4 Probesets Monitor a Simple Cassette Exon

  26. Some Examples

  27. Highly Conserved 12bp Exon

  28. CLASP1 Event #1

  29. CLASP1 Event #2

  30. Excel Sheet with Combined Results

  31. 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

  32. 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

  33. Histogram of Cassette Exon Size 16 Exons < 25 bp

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