1. Summer 2005 Show Me the CpG Islands! Alicia Laughton (Mathematics ‘06) Jessica Minnier (Mathematics ‘07) Guided by Yung-Pin Chen (Mathematics/Statistics) (With Statistical Significance)

2. Summer 2005 This work is funded by John S. Rogers Science Research Program

3. Summer 2005 Outline DNA Overview CpG Islands Methods –Traditional Method –Our Method Future plans

4. Summer 2005 DNA Deoxyribonucleic acid Double-helix Chain of nucleotide subunits Contains genetic information

5. Summer 2005 Nucleotides Made up of sugar, Phosphate, and bases Four bases –Adenine (A) –Cytosine (C) –Guanine (G) –Thymine (T) CpG represents a C directly followed by a G in the DNA sequence

6. Summer 2005 Methylation Causes C to turn into T Accounts for low occurrence of CpG dinucleotides in vertebrates –Expectation is 6.25% randomly –Actually 1% of total sequence (Bird 1986)

7. Summer 2005 Sequence AL031723 Human DNA sequence on chromosome 16 3 known CpG Islands Percentage of Content: –A - 22.7% –C - 29.5% –G - 28.3% –T - 19.5% –CpG - 3.1%

8. Summer 2005 CpG Islands “regions of DNA with a high G + C content and a high frequency of CpG dinucleotides relative to the bulk genome” -- Gardiner-Garden and Frommer (1987)

9. Summer 2005 CpG islands & Genes Gene 5’ end CpGi Gene Promoter CpG islands Gene CpG islands in body Gene 3’ end CpG islands

10. Summer 2005 What is important about CpG Islands? Useful in identifying protein-coding regions (Yoon and Vaidyanathan, 2004) –Associated with “housekeeping genes” and 40% of tissue-specific genes Aberrant methylation of CpG sites may cause silencing of tumor-suppressor genes (Deng, Zhou et al, 2002)

11. Summer 2005 aggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagtgtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttgagacggagtcttgctctgtca cccaggctggagtgtagtggtgcaatctcggctcactgcacctctgcctcccgggttcaagcgattttcctgcctcagcccccggagtagctgggattacaggtgcccgccaccacacccagctaatttttgtatttttagtagagacggggtttcgccatgttggccaggctggtctcgaactcctg acctcaggtgatccgcctgcctcagcctcccaaagtgctgggattacagacgtgagccactgcggctggcctctctccccgtctttaactgtagccctgtgaattctcatcagcctgggcctggactcagcaggccaaaaagttaccagcagagcccagcacatgtgaggaaagtcggagacgt ggcggcgccggccggaggatccttcccaagaccctgggccgctgtggccccctagatcttgcaggttgccagggtgccaggccagggagggggcctttctgagattctcctcattctgacacaggagaggagggcactgacccagtcccaaggtcccgggggaatcagccgaccacagc ccaggactgtcccacctgggcagagagcccattctgggtgcccagcccgggcaggcccaggcacccccagcagtgccccgggcagcacctgccagccaggtagtgcagggtgaggttgggcagggcagggcgtggtaggtcagctgagcaaacagctcggagggagagctgggga gggctgggaactaggtcgatagaaacacagggactgtgttagggaggggatgccttgccagtcacgcccagccctgactcctgccctctgagggggcttcccccacccctgctgacagccccaggaccggcccctgccaggaggctgacctgccaggagtgaccgccccagacttgagc ccttgggaggcaggttctgagtccccttttcctgctcagacccccagggaaacgcaggctgggccagaggcagctgcacagacccctgcagtggggtgctcggtggagagcgctggaggtgggagggaggatgtgtgaggcagcgggagagaatccaggcttcccccacaacacccac catgagcggtgcagagtaggggtgggcggcacgggagccttcccaccccgcagaaccaggccctgggcagagctggcctacagacgataccggacaagtcctcctccgtcttggtgacagagggagctgggactccctccacccacccactgccacttcagaagcagccacagggaga ctgggaggggcaggggtgctggggatgagcgtggggctcagccctccctcttcccaccctggagggctgcctccttccagcccacctggaagggtggtgtcagtcccagagcccctgcactccccgccccacctcctgcagctggaacccgcgtgggagccgcacccagcgtcccaggg acaaacacagaggccttgggtggtggcggtaccaaggtctgaggcctggcagctcaggggcacccccgtccctgagagaggtcaagaaggggaggcaccaccccccaccacgggacctcgctgacgatgcccatagagagaaaccaggccagtgctgggaggggaaagaccccag gcctcatgagaagtcactgcctgcttttcccctcggccaggaaggaagccccaggcccttccctcccgtctcgggcatactgaccccaggcaccaagcgagaccaggagcccacccctttcctttcccagatggcacaccagtgactctgaatatcggagcgcacccctgctccctgggaggc aggatatcgtgccgctgctccctggggcgcacgataccctccccaggaaggcgccggtcagggcggacgggccagggtgctcaccggtaccaggcgaggccgcgctcgtagcacctgtcgaagaagtggggctcagagcccagcgcgcggacgtcggggtgcagccgcagaaactc cagcagggcgcgcgtgccgcccttcttcacgccaacgatgagcgcttgcgggaagcgccggcggccgggaccgctggccaaaggcaggccgggtgctcccgggcggtggacggagctggacggctcggagggcgcgggggccggcgcgggggcgcgggcggccggcgggca gcggccggggagggcgcagaggcagtaggcgccgagcaccagggccacgagcagcatcggcgcgcgggacgcccgcagagcggccccttgcccggcccctgcgccctggccgcccccggccccgccgcccaggccgccgctacctgccatggggtcgcgccgctccaggccc gggagcgggggcagcaggcgggcgcgcatctcggcccgcgcgccgctcagtccgtgggtgcccggcttgtgctctgcgcccggcggtcccgcagcctgggagcgggcgcggggcgggaccgggggcggggtctggacgccctcccccctccccctcccccgcccactccgcctcc gaggccactgcctgggctggacccgccggcagccgccaccacccgggcgcgactcgagctgccgggaccaccaggacgctcctgctccgagatcccaggccctggctcgcttgactccggcatcttcacctctgcgcggggaggatgcggcggcggtggccgttcgggacgcagggc agggacagggcggcgcgcgggcctcgggaccctctgtttgaagaccgatccccttccccccccaccccactccgggacgtgcgcggcaggtgcataggccaagccttggcctgcaggagcgggagcctcatcgccaggccaaggggacccaggaaaagcgtcgatccgggcactcg gcctgccaagggagaaagaggccgggacagcaccctagtgtgcagagagggatcccagaacgtgtggggggagtctgcggccgggaatggcgtgcgcctcctcttcctgcctgctggagggaccagcaccaaaacaggaaagttcaccctgccaggccttctctccaaagagtcagag ggagctccgtagggggatggggttcccggaccccctgccgtggaaggggagtgggaacacagacaggcggcaagggctttcgaggccccctcttgcacaaaccagctcagagatcggagatctttgggatcaattactttccctccccaggcatccgaagcctatcctagcccaggtgtgg atgagggtgggagagacgggggaggagggagaggagcaggactggacccccgtgtgacaaacatctgacaagttgctctgaggactgcccccctccttgtggagcccacctcatctggtgtgcatttccctgcggctttcatccagccctgggcgaccctccctcctccatctcagcctccct cctcctgccccacacctcaggcctgggactcgcagatgccaaaagggcctggcagatgccaaagccagaaagtgcagggggactgcatcccccacaggagaccgggttcttccccactacatactcagaccccactccctgcacccactgctcttgcaaaccaggaactaaggggttcccc tacccaccccgctccttgcctcctcttgcttttcttttgttttgtttgtttttgagacagagctgcactccagctgactcttgtcgcccaggctggagtgcagtggcacaatctcagctcactacaacctctgcctcccgggttcaagcgattctcctgcctcagcctcccaagtagctgggaatacaggca cccatcaccacgcctggctaatttttgtatttttagtagagatggggtttcaccatgttagtcaggctggtctcaaactcctgacctcaggtaatctgcccacctcagcctcccaaagggctgggattacaggcgtgagccactgtgccccaccctcctcttgcttttctaaaagatgatggtcaaagtac agcccccatttgcccccagacagggcacccttcccagatcgagaccttggggagtctgcgtgacccccacacctggcagacacaggtgcttcactagtgggggaacggctgagcatgtgctgagctcgggggcactagtgggctacagtccccaagtgggaggcccctcaagagcctgga tgagctgactgacggtggagaggagggaaggagggcctatggccaaagtcaatccaggacccaactgccgaggccacaggaaggccgggtcaccgcctggaactaggtcggtcacagcccagtgggagccgtggcccggagactcaactgggggccctggttactctgctcgcctccc cgcgtcggcacccagaacagagcttgcaggcactgggggcccagtccagggtctcaagagcagacaatgctgccttgcagttggggaaactgagacagggtgagaactttcagaggctcattgcaggctcctagcaggctgaaaggacggaggcacaggcacctaggagcacaccagc cccacgtggccacggcccctcggagagcatgaggacacttgcaatgcggaagctcagcaggcccagctctactggctctgcaccgcccagtgaggggtcagcacagttggtccaagggacaataccagattaatgaggcagaagccacgggactgaccccttggaattctccacacccac actgtgcatccttaacccaaagcttctagcttggtagcccctcctaccctcctccctgcagcagggattagggatgcattctgacccctgcctgccgtcaggggagtgaggtctctccctggagcctgagctgaggatgcccaattcagccaggtgagccccgggatggactccatgtcccctag ccaccacctgacttccccagcaccccacactggcaccagcccttcagatctcagaagcgagccaccctattctcacggagccccttcctgcctgccctccaaacccaagagtagttttagtacaaaaggcaaagttaacaaataggggtaggcgtcagggaaggaagaggatcagaggatcg ggaacggagaaactggagcacctggagaagcgtctgggtcctgccacccccactgactccccaactggccttgggcagggtcctctctgcaggcgctgggtccaagcttggggatgagcagccaccagcgcgggctgcttcagctgaggctgccgcacccccacgtccatcctgggtaga ggcaggacagccacagagccccatgcacggggctggactcaccctgggcactcacctaaaggcagtctcctcctttccaaagcccagactttctccggactcccaggaccaccaacaagggttcctgtgcgcagactcgggggtcttggggaggaaggacgctttctaggtggctgcctgg aacctggaggcccctttctacagtacctggccagcggtcggtcacacctgagtgcccagagtgagcgggcggcagaggcatttctgacgctgccaggtaatcccacgggctggaaacgacctctgggctgggaagccaccgcctcccccagtcctgctgggtccctcagcagagagaacg gaaccggggctttccccacagttttcaaagtttcagggaatcctagccaagtatcattccttcttccggagccgggaccccaggtcaagcctggggcccccacagggcggtcccaaccccactgcccggagcgcacccctgctccctgggaggcaggatatcgtgccgctgctccctggggc gcacgataccctccccaggaaggcgccggtcagggcggacgggccagggtgctcaccggtaccaggcgaggccgcgctcgtagcacctgtcgaagaagtggggctcagagcccagcgcgcggacgtcggggtgcagccgcagaaactccagcagggcgcgcgtgccgcccttcttc acgccaacgatgagcgcttgcgggaagcgccggcggccgggaccgctggcgtttccctcccaggggcccagtggtgaactgaattcaggcctgagacatactctgtctactaagtcaccccatctgcccagccttggtccacctggcactgcccagagacatcagtgatgcatttcggaagct ggcaaagtggaccccactggagtacaaaggactcagggacccctgtgctggggaagagaaggagcccaggacctcccccaggggctgcctctgaggggcgtgagattcaggggcctctcgggtgggacctgcgggggccgctagacactgcgggaacttcacatccccaacgcccag cagcagcctgcagggaaggcaggggaggcgagccgggctcagagagggcgagcaacttgccccatccgaaggcaaaggtggtatgagacccgggtcctctctccacctctgccccagccttcctggccacagggctggcgccaggcaggcacggcacaggctcccggcagaggcc acggtctcagccatccccacggtctcaggagtccccacggtctcagccgtccccacggtctgagtccccacggtctcagctgttcccacggtctcaggagtccccacaggttcagcagtccccacggtctcagccatccccacggtctcagccgtccccacagtctcagccatccccacggtct cagcagtccctactcaggacttgaaattccagcactggttccgtgatggctcctccagccccctgcccagcccagcatggtcatttccatctcctggcctttccgctgccgtctctctgctggatgctttatccttagtccccgctgagggcagaaggactttccaggaggaattgaccagaacgcag aacagcaggatgtggaatggactggggacagggagagagagatgcagggaccaggagtcggctcggagggttctcctggaagctgacccctccctccatcaggcactcggctgacggtggctacacacctcggggcgcccaggatggcagcactggggctgttcattcaccagtggatc cccagcacctaacagagcctggcacgcagtggacattccattaatgtcgctcagtggaagggtatacgtgggaggagaggtcgggaaggctttctggaggtgacggccaggtgaagacgaggagaacagcattccaggccaaggaaccgtgtgggtgaaggctcagcagcagagagcc cgggcagtagaggatggggtggagcttaaggccctgcgggaacaggggcggggcttagagtctggcctgaggctggtccagccccgcctcctcctcaggctcccaccaactctgagccaccagaccctcctttgtaaaatgaagacctcagtcatgactcgcatgagtctctgaagagtaac agctttattgtgatgtaattcacacaccactcaatccagccatttgtcgcatgcaaatcaatggttttcagtatattcatagtcgtgcaatcacaatcaattttagaacatttctatcaccccaaaaagaaatcctgtgtccattagcaatgacgccctcttctccccttcccacagcccctggcaaccacgaa tctactttctgtctctatgggtttgcctattctggacatttcacaaaaagagaatcattgcttgaagccaggagttcaagaccaacctgggcaacaaagcgagaaccccgtctgtacaaaatattttaaatttagccaggcacagtggcgcacaccagtagtcccagcactttggaagtctgaggcagg aggttcacttgaggcggggaattcaaaaccagcctgggcaacatagggagtaccagtctctacaaaaaatttcaaaatttgccaagcgtgatggtatgcacctatagtcctagcttactcaggaggctgaggtgggaggatcgcttgagcccaggagtacgaggctgcagtgagccatgatcata ccactgcattccagcctgggcgacagagtgagagcccatctctaaaacagaaagaaagaaagaaagaaatatggccagtcacagtggctcatgcctgtaatcccagcattttgggaggccaaggcaggtggatcacttgaggtcaggagttcgagaccagcctggccaacatggtgaaacc ctgtctctaccaaaaatacataaattagccaggtgtgggccaggcgccatggcttacacttgtaatcccagcactttgggaggccgaggtgggcagatcacctgaggttgagagttcgagaccagcctgaccaacatgaagaaaccctgtctctactaaaaatacaaaaaattagctgggtgtggt ggtgcatgcctgtaatctcagctacttgggaggctgaggaaggagaatggcttgaacccgggaggcagaggttgtggtgagccgagatcgcgcgattgcactccagcctgggcaacaacagcaaaactccatctcaaataataataataataaattagccaggtgtggtggtgcacgcctgtag tcccagctactcgggaggctgaggcacaagaaacccttgaacccgggaggcagaggttgcagtgaagctgaaattgcaccattccactccagcctgggagacagagtgagacaccatctctaaaatgaaaaaaaaaaaagagaatcatacaatgttcgtccttttgtgtctgggtctcttactca gcatgttctccaggttcatcaacactgtggcatgtgccagtacctccttcctcttcctgactgagtaatactccatcgtatggatggaccaccttttgttgattccctcattcgttgatggacatctaggttgtttccactgcggggttcttagtaacggtattacagggaaccatagattaccaggtatt How do you locate the CpG island in a DNA sequence?

12. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg C+G Content: 0.492 Observed/Expected: 0.548

13. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg C+G Content: 0.501 Observed/Expected: 0.568

14. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg C+G Content: 0.500 Observed/Expected: 0.560

15. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg C+G Content: 0.712 Observed/Expected: 0.604 200 steps later…

16. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg C+G Content: 0.598 Observed/Expected: 0.421 600 steps later…

17. Summer 2005 Just a couple formulas… G+C content = (# of C’s) + (# of G’s) length of window Obs/Exp ratio = Observed # of CpGs # of CpG’s in window Expected # of CpGs (# of C’s)x(# of G’s) length = From window

18. Summer 2005 Traditional Methods Gardiner-Garden and Frommer (1987) –Window size 100 bp and Shift size 1bp –Criteria At least 200 base pairs G + C content greater than 50% Expected portion of the Obs/Exp ratio calculated over the window Obs/Exp ratio greater than 0.6 Takai and Jones (2002) –Window size 200 bp and Shift size 1bp –Criteria At least 500 base pairs At least 7 CpG dinucleotides in 200 base pair sequence G + C content greater than 55% Obs/Exp ratio calculated in same fashion as above method Obs/Exp ratio greater than 0.65

19. Summer 2005 The Traditional Method C+G content Obs/Exp ratio C+G content/Obs-Exp ratio Base Position Sequence AL031723

20. Summer 2005 Modifying the traditional methods –Window size 200 bp and Shift size 1 bp –Expected portion of the Obs/Exp ratio is based on whole sequence And…. Our Method Observed # of CpGs # of CpG’s in window Expected # of CpGs (# of C’s)x(# of G’s) length = From entire sequence

21. Summer 2005 Cutoffs greater than 97th percentile of observed sequence Obs/Exp Ratio G+C Content Mean:0.0018 Standard Deviation:0.0014 97th percentile:0.0058 Mean:0.5815 Standard Deviation:0.0818 97th percentile:0.7350 G+C ContentObs/Exp Ratio Number of Observations Sequence AL031723

22. Summer 2005 Kullback-Leibler Divergence p ln (p/0.03) + (1-p) ln ((1-p)/(1-0.03)) p KL Divergence

23. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg Kullback-Leibler: 0.508 Our Obs/Exp: 0.0029 C+G: 0.492

24. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg Kullback-Leibler: 0.509 Our Obs/Exp: 0.0030 C+G: 0.501

25. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg Kullback-Leibler: 0.507 Our Obs/Exp: 0.0029 C+G: 0.500

26. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg 200 steps later… Kullback-Leibler: 0.520 Our Obs/Exp: 0.0033 C+G: 0.712

27. Summer 2005 agaattgcttgaaccgggaggcggaggttgcaatgagctgagatcacaccactgcactccagcatggtgacagagcaagactccatctc aaatcgagtaaaaaaaaaaaaatagctgggtgcggtggctcacgcctgtaatcccagcactttgggaggctgaggcgggtagatcacga ggtcaggagatcgtagccatcctggctaacacggtgaaaccccgtctctactaaaaatacaaaaagaaattagctgggcgtggtggtggg cgcctgtagtcccagctactcgggaggctgaggcaggagaatggcgtgaacccgggaggtggagcttgcagtgagtcgagatcacgcc actgcactccagcctgggcgacagagcgagactcgatctcaaaaaaaaaaaaaaaaaaaaagtgcgacacgaggcacacagtcagtg cccagtggagttcgctgatatggttaccacatccctggggacagcgcctccaccctccaacctcgaggtttgtggaaaaatctgggtccaa gctttatttcttaaatattcctctctgcccagcatgtgcacgcagcccgctctggccaggcgagcgggtgtcaatcaaggtgctgagcatccc cagggtgccgctcagccccagccgaagtcctggcccgtcatctggtagaacctgcggttgaagggccggtagaactcctgcaggcgcc ggaccagggcctggggcacgcgtgggtgtggccggcccttggacttgcccaggcagcggggacggctgccgccctgggccttcttga ggcaggggaagcccttggtggcgttgaagtagaagtgcttgtccgtgacgacccgtttcaggcccaggaagtcctgcacgcggccgacc tctccggccgggtcgctgaccagacgctccccgctgacgaacaggaagtgggacagggggaagtagcgcagccagtggtccaggtgc tgggcgtacaggccgatgcggacggcgctccaggctgtgtccacggggcccaggccgtggcggaaggccagggcgcggaagctgg gcaggcccggggtcttggagagcgtctgggcgtagtcggagatggcccgggtcacggggttccgcaccaccacgatcagcttcgtgtc cggggacatggcgtggatgcggcggggggcctctcgcgtcacgaagtagctgggggtcttctccatggtgatctgcccatccagggttc ggggcatcagactcctgcgggacgggtgcaaggagagggggcctgagcctccccagccctagaccggcccccaggggcccgggac caaggcccccttatgcccgggaagcccaggcctccagggcgagcaagtcttcctccctgctcgggcccacccctgctagcgtgcgcgg ctgggcagcctggaacatggactgtgagggtgcccagcccggcacctgcctgcagcccggcctgttccgccggcctgccccgcctgct gctgcactgaggattagggtgacggtcgctggtcgggaggcccaaatgctcctcaccacccacatatcttccctgtgcaatccctgccgtc ctcgcttccagagccagctccctcccaccggacccacactttcctggaactaggctgcccccagctcctttctcatcccagaccaagtaccc cgaggcccgcccgcctagatcacttgaggtcacccgttcactcagtggctgacagcatcccctaaatcagcccttcaccaattattgacagt gtgtcctcaaccaaaagtagtcctccctgctccctccctcccctgatgtaattacatctcttcccatctttatttattttttg 600 steps later… Kullback-Leibler: 0.510 Our Obs/Exp: 0.0030 C+G: 0.598

28. Summer 2005 Our Method KL Divergence*12/Obs-Exp ratio*160/C+G Content Base Position Kullback-Leibler DivergenceObserved/Expected RatioC+G Content Sequence AL031723

29. Summer 2005 Comparison of AL031723 Traditional Method Our Method

30. Summer 2005 Comparison of AL031723 Traditional Method Possible CpG Islands 3878-45345849-6136 6541-68208479-8698 10745-1104918435-19580 25131-2635935182-35441 36245-3657636827-37606 Actual CpG Islands 18928-19547 25201-26371 36997-37693

31. Summer 2005 Comparison of AL031723 Our Method Possible CpG Islands 19227-19435 25197-26147 36982-37420 Actual CpG Islands 18928-19547 25201-26371 36997-37693

32. Summer 2005 Cons Traditional Method –Criteria not stringent enough –If the expected part of the Obs/Exp ratio is unusually high then a high CpG count may not bring ratio above the cutoff Our Method –Criteria sometimes too stringent

33. Summer 2005 Future Plans CpG Islands Linkage Disequilibrium and SNPs –Statistical analysis of the linkage disequilibrium coefficient –Kullback-Leibler Divergence II

34. Summer 2005 Thank you! Former researchers –Andrew Dittmore –Yasuhiro Goda –Nick Heppenstall –Michal Dvir Deborah Lycan John S. Rogers Program