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Discovery the Relationship Between Single Nucleotide Polymorphism and Alternative Splicing events

Discovery the Relationship Between Single Nucleotide Polymorphism and Alternative Splicing events. 逢甲大學 資訊工程系 / 生物資訊研究中心 許芳榮. Outline. 1. Object 2. Introduction and background 3. Materials 4. Method and result 5. Conclusion. Object.

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Discovery the Relationship Between Single Nucleotide Polymorphism and Alternative Splicing events

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  1. Discovery the Relationship Between Single Nucleotide Polymorphism and Alternative Splicing events 逢甲大學 資訊工程系/生物資訊研究中心 許芳榮

  2. Outline • 1. Object • 2. Introduction and background • 3. Materials • 4. Method and result • 5. Conclusion

  3. Object • We combined the data from available dbSNP and alternative splicing database AVATAR to find out the relationship between SNP and alternative splicing.

  4. Introduction and background

  5. SNP SNP The genetic polymorphism due to base substitutions is called single nucleotide polymorphism (SNP) A: ATTCGC B: ATTAGC

  6. SNP Classification

  7. RNA Splicing

  8. Alternative Splicing Classification

  9. Materials • SNP Database • Alternative splicing Database • Align ESTs to genome

  10. SNP Database • dbSNP database (Build 120 , Mar 18, 2004) ftp://ftp.ncbi.nlm.nih.gov/snp/ • 9098790 refSNP sequences (RS SNP)

  11. Alternative splicing Database • A value added transcriptome database (AVATAR) • Contain 174,546 of human alternative splicing events

  12. A value added transcriptome database

  13. Align ESTs to genome • Mugup use the multi-layer unique makers alignment method

  14. ESEfinder

  15. Method and result • Finding exonic SNP dependent alternative splicing event • Analyzing alternative splicing related SNPs

  16. Finding exonic SNP dependent alternative splicing event • Finding exonic SNPs on the human genome • Finding EST supported exonic SNPs and polymorphic nucleotides • Finding candidates of exonic SNP dependent alternative splicing event • Confirmation of candidate exonic SNP dependent alternative splicing event

  17. Finding exonic SNPs on the human genome AVATAR exon intron exon dbSNP We find out 223,010 exonic SNPs

  18. Finding EST supported exonic SNPs and polymorphic nucleotides • We use Mugup to align all ESTs (5406242) to human genome • Discriminating the SNP and polymorphic nucleotide

  19. Mugup Genome EST 1 EST 2 EST 3 EST 4 EST 5 We align 3,400,000 ESTs to genomr and we get 12,000,000 mismatch

  20. Discriminating the SNP and polymorphic nucleotide Genome Have refSNP data No refSNP data and only 1 EST supports No refSNP data and over 2 ESTs supoort EST supported polymorphic nucleotides (478,461) EST supported exonic SNPs (241,722)

  21. Finding candidates of exonic SNP dependent alternative splicing event • Finding candidates of exonic SNP dependent alternative 3’ splicing site event and alternative 5’ splicing site event • Finding candidates of exonic SNP dependent retained intron alternative splicing event • Finding candidates of exonic SNP dependent alternative cassette alternative splicing event

  22. Finding candidates of exonic SNP dependent alternative 3’ splicing site event and alternative 5’ splicing site event Alternative 3’ splicing site event Alternative 5’ splicing site event

  23. A. B. C. D. Exon Alternative splicing exon SNP Intron

  24. A. B. C. D. Exon Retained intron SNP Intron Finding candidates of exonic SNP dependent retained intron alternative splicing event

  25. A. B. C. D. Exon Cassette exon SNP Intron Finding candidates of exonic SNP dependent alternative cassette alternative splicing event

  26. Confirmation of candidate exonic SNP dependent alternative splicing event • Fisher’s exact test • Confirmation of exonic SNP dependent alternative 3’ splicing site event and alternative 5’ splicing site event • Confirmation of exonic SNP dependent retained intron alternative splicing event • Confirmation of exonic SNP dependent cassette alternative splicing event • More than one SNP support alternative splicing event

  27. Fisher’s exact test

  28. Confirmation of exonic SNP dependent alternative 3’ splicing site event and alternative 5’ splicing site event a. Polymorphic nucleotide (SNP) with alternative splicing form. b. Polymorphic nucleotide (SNP) with normal splicing form. c. General nucleotide (nucleotide on EST as genome) with alternative splicing form. d. General nucleotide (nucleotide on EST as genome) with normal splicing form.

  29. SNP_RS GENE Nucleotide type (genome/est) CONTIG_GI POSITION SNP LOCATE ALTERNATIVE SPLICING EVENT P VALUE 10840159 LOC283120 A G 37541814 780864 D 3S 2.87E-57 4968215 EIF4A1 G C 37543500 6318943 D 3S 1.28E-41 2839702 LOC283120 A C 37541814 780957 D 3S 1.61E-33 1049750 CCL4 G T 37544509 9168267 U 3S 6.40E-29 7216854 CCL4 G A 37544509 9168341 U 3S 1.24E-18 9635771 CCL4 A G 37544509 9168312 U 3S 4.34E-18

  30. SNP_RS GENE Nucleotide type (genome/est) CONTIG_GI POSITION SNP LOCATE ALTERNATIVE SPLICING EVENT P VALUE 1803621 GAPD T C 29805200 6501370 U 5S 3.63E-41 1042178 HLA-DPA1 T C 29804415 23834681 U 5S 1.96E-37 1719146 CCL4 T C 37544509 9168941 U 5S 1.07E-32 6926 PTGDS A C 29793214 627220 D 5S 2.35E-24 1650294 LDHB A G 37543832 14556003 U 5S 4.76E-24 6520277 TIMP1 T C 37546162 3854375 U 5S 8.63E-19 9280793 HLA-A G A 29804415 20768511 U 5S 3.49E-18

  31. Confirmation of exonic SNP dependent retained intron alternative splicing event a. Polymorphic nucleotide (SNP) with retained intron form b. Polymorphic nucleotide (SNP) with intron splicing form. c. General nucleotide (nucleotide on EST as genome) with retained intron form. d. General nucleotide (nucleotide on EST as genome) with intron splicing form.

  32. SNP_RS GENE Nucleotide type (genome/est) CONTIG_GI POSITION SNP LOCATE ALTERNATIVE SPLICING EVENT P VALUE 962004 ALB C T 29791409 2792249 U INTRON 1.25E-13 2076172 RPL10A C T 29804415 26234052 U INTRON 1.74E-09 4234491 PCNP C T 37550867 7790730 D RETAINED INTRON 4.54E-09 7901604 ECHS1 G A 37550477 339705 D INTRON 3.80E-08 1071726 UBC A G 37543915 2706898 D INTRON 3.69E-07 7137123 UBC A G 37543915 2706903 D INTRON 3.69E-07

  33. Confirmation of exonic SNP dependent cassette alternative splicing event a. Polymorphic nucleotide (SNP) with exon form. b. Polymorphic nucleotide (SNP) with exon skip splicing form. c. General nucleotide (nucleotide on EST as genome) with exon form. d. General nucleotide (nucleotide on EST as genome) with exon skip splicing form

  34. SNP_RS GENE Nucleotide type (genome/est) CONTIG_GI POSITION SNP LOCATE ALTERNATIVE SPLICING EVENT P VALUE 1049804 CCL4 T C 37544509 9169628 D EXON 1.99E-09 1049805 CCL4 T C 37544509 9169634 D EXON 1.99E-09 1049807 CCL4 A G 37544509 9169643 D EXON 1.99E-09 1049819 CCL4 C T 37544509 9169791 D EXON 8.96E-09 1049821 CCL4 T G 37544509 9169803 D EXON 8.96E-09 1049823 CCL4 T C 37544509 9169810 D EXON 8.96E-09

  35. Result

  36. More than one SNP support alternative splicing event Have similar ESTs support (90%)

  37. 2 Alternative splicing NO. C20orf98 Gene Alternative splicing event e Cassette Exon Skip 27501067 Contig_gi Position 274201 Nucleotide type (genome/est) C A Orient 0 SNP locate D First intron start 270008 First intron end 270281 Second intron start 270477 Second intron end 273853 2 C20orf98 Cassette Exon Skip 27501067 274535 C A 0 D 270008 270281 270477 273853 3 MRPS2 Cassette Exon 29731701 1111075 C A 0 D 1109285 1109379 1109578 1110852 3 MRPS2 Cassette Exon 29731701 1111126 C A 0 D 1109285 1109379 1109578 1110852 Totally we find out 5290 alternative splicing events which have more than one SNP or polymorphic nucleotides may influence on it

  38. Analyzing the alternative splicing related SNPs • Analyzing the SNP location in exon • Analyzing the sequence in alternative splicing related SNPs area

  39. Analyzing the SNP location in exon Average distance form nucleotide to alternative splice site is 114.2 bp

  40. Sliding window (window size 6~100) Seq 1 Seq 2 Seq 3 : SNP 201 bp Analyzing the sequence in alternative splicing related SNPs area

  41. Consensus sequences ESTs support GNCCAAG 19 CCAAAG 17 AAAGNTG 16 GGNGCCC 16 AAAAAG 16 ANAAAGA 15 Totally we find out 1489 consensus sequences of different alternative splicing event related SNPs areas

  42. ESEfinder The 72% (88/122) of the sequence have the binding site variation when the SNP changed.

  43. Conclusion • We combined the data from available dbSNP and alternative splicing database AVATAR to find out the relationship between SNP and alternative splicing • Through our study method, we can quickly find the possible candidates related SNP and alternative splicing event

  44. We can find the possible relationship between human alternative splicing changes and SNPs rapidly, it may promote the advances of biotechnology • There are more than one SNPs to influence on same alternative splice site

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