1. SAGE data in StemBase Christopher Porter Ottawa Health Research Institute

2. Presentation outline SAGE protocol SAGE analysis Integration with Affymetrix data Access to SAGE data in StemBase

3. Basics of SAGE Identification and quantitation of mRNAs in a mixed population by generation of a (usually) unique sequence tag. Assumes that tags are generated in proportion to mRNA abundance in the population

5. CTCTAGATGCATGGTTCTCATTTTTTGAGGTTGAAAAGTGGCTTTACATGGTGG CTCACAACCATCTGAGCCCTGGCTCTGTCACATGTTAATATTTAATTAGAGAAA TCACACTTCCCACATGTTTTATTTATATTCAAGCATCCCCGGCTGTCCCATGCT CGAGTTTCTTCCTGTGATATATCTCTCTTCACATGTCTGGAGACAGTAGGGGCA CATGGTGGCTCACAACCATCTGAGCCCTGGCTCTGTCACATG CATGTTAATATTTAATTAGAGAAATCACACTTCCCACATG CATGTTTTATTTATATTCAAGCATCCCCGGCTGTCCCATG CATGCTCGAGTTTCTTCCTGTGATATATCTCTCTTCACATG GTGGCTCACAACCATCTGAGCCCTGGCTCTGTCA CACCGAGTGTTGGTAGACTCGGGACCGAGACAGT TTAATATTTAATTAGAGAAATCACACTTCCCA AATTATAAATTAATCTCTTTAGTGTGAAGGGT TTTTATTTATATTCAAGCATCCCCGGCTGTCC AAAATAAATATAAGTTCGTAGGGGCCGACAGG CTCGAGTTTCTTCCTGTGATATATCTCTCTTCA GAGCTCAAAGAAGGACACTATATAGAGAGAAGT Sequence to Tags

6. GTGGCTCACAACCATCT TGACAGAGCCAGGGCTC TTAATATTTAATTAGAG TGGGAAGTGTGATTTCT TTTTATTTATATTCAAG GGACAGCCGGGGATGCT CTCGAGTTTCTTCCTGT TGAAGAGAGATATATCA +-------------------+----------+ | tagSeq | tagCount | +-------------------+----------+ | CTCGAGTTTCTTCCTGT | 58 | | GGACAGCCGGGGATGCT | 1 | | GTGGCTCACAACCATCT | 461 | | TGAAGAGAGATATATCA | 3 | | TGACAGAGCCAGGGCTC | 6 | | TGGGAAGTGTGATTTCT | 92 | | TTAATATTTAATTAGAG | 56 | | TTTTATTTATATTCAAG | 2 | +-------------------+----------+ Library database

7. SAGE tag identification Match to tags predicted from known sequences –e.g. SAGEMap –Generate mappings from cDNA sequences

8. Finding tags for a gene >gi|7305398|ref|NM_013633.1| Mus musculus POU domain, class 5, transcription factor 1 (Pou5f1), mRNA GTGAGCCGTCTTTCCACCAGGCCCCCGGCTCGGGGTGCCCACCTTCCCCATGGCTGGACACCTGGCTTCA GACTTCGCCTCCTCACCCCCACCAGGTGGGGGTGATGGGTCAGCAGGGCTGGAGCCGGGCTGGGTGGATT CTCGAACCTGGCTAAGCTTCCAAGGGCCTCCAGGTGGGCCTGGAATCGGACCAGGCTCAGAGGTATTGGG GATCTCCCCATGTCCGCCCGCATACGAGTTCTGCGGAGGGATGGCATACTGTGGACCTCAGGTTGGACTG GGCCTAGTCCCCCAAGTTGGCGTGGAGACTTTGCAGCCTGAGGGCCAGGCAGGAGCACGAGTGGAAAGCA ACTCAGAGGGAACCTCCTCTGAGCCCTGTGCCGACCGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACC AACTCCCGAGGAGTCCCAGGACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCCAAGCTGCTGAAG CAGAAGAGGATCACCTTGGGGTACACCCAGGCCGACGTGGGGCTCACCCTGGGCGTTCTCTTTGGAAAGG TGTTCAGCCAGACCACCATCTGTCGCTTCGAGGCCTTGCAGCTCAGCCTTAAGAACATGTGTAAGCTGCG GCCCCTGCTGGAGAAGTGGGTGGAGGAAGCCGACAACAATGAGAACCTTCAGGAGATATGCAAATCGGAG ACCCTGGTGCAGGCCCGGAAGAGAAAGCGAACTAGCATTGAGAACCGTGTGAGGTGGAGTCTGGAGACCA TGTTTCTGAAGTGCCCGAAGCCCTCCCTACAGCAGATCACTCACATCGCCAATCAGCTTGGGCTAGAGAA GGATGTGGTTCGAGTATGGTTCTGTAACCGGCGCCAGAAGGGCAAAAGATCAAGTATTGAGTATTCCCAA CGAGAAGAGTATGAGGCTACAGGACACCTTTCCCAGGGGGGGCTGTATCCTTTCCTCTGCCCCCAGGTCC CCACTTTGGCACCCCAGGCTATGGAAGCCCCCACTTCACCACACTCTACTCAGTCCCTTTTCCTGAGGGC GAGGCCTTTCCCTCTGTTCCCGTCACTGCTCTGGGCTCTCCCATGCATTCAAACTGAGGCACCAGCCCTC CCTGGGGATGCTGTGAGCCAAGGCAAGGGAGGTAGACAAGAGAACCTGGAGCTTTGGGGTTAAATTCTTT TACTGAGGAGGGATTAAAAGCACAACAGGGGTGGGGGGTGGGATGGGGAAAGAAGCTCAGTGATGCTGTT GATCAGGAGCCTGGCCTGTCTGTCACTCATCATTTTGTTCTTAAATAAAGACTGGACACACAGT 0 1 2 3 4 5

9. Tags in database +-------------------+------+-------------------------------------------------------------------------+ | tagSeq | rank | geneName | +-------------------+------+-------------------------------------------------------------------------+ | CATTCAAACTGAGGCAC | 0 | Mus musculus POU domain, class 5, transcription factor 1 (Pou5f1), mRNA | | TTTCTGAAGTGCCCGAA | 1 | Mus musculus POU domain, class 5, transcription factor 1 (Pou5f1), mRNA | | TGTAAGCTGCGGCCCCT | 2 | Mus musculus POU domain, class 5, transcription factor 1 (Pou5f1), mRNA | | AAAGCCCTGCAGAAGGA | 3 | Mus musculus POU domain, class 5, transcription factor 1 (Pou5f1), mRNA | | TCCGCCCGCATACGAGT | 4 | Mus musculus POU domain, class 5, transcription factor 1 (Pou5f1), mRNA | | GCTGGACACCTGGCTTC | 5 | Mus musculus POU domain, class 5, transcription factor 1 (Pou5f1), mRNA | +-------------------+------+-------------------------------------------------------------------------+

10. Finding tags in genomic sequence >1 dna:chromosome chromosome:NCBIM36:1:4729550:4729591:1 GAAACTGGCTCAGTGTAGCCATGAAGTCCAGGCCACTAACCT CTTTGACCGAGTCACATCGGTACTTCAGGTCCGGTGATTGGA |||||||||||||||||||||||||||||||||||||||||| 24,837,922 tags generated Tags observed at 1 - 36,168 locations 96% of tags are from a single location

11. Associating tags with probesets

22. UCSC Genome Browser controls

25. Conclusion Please contact ogicinfo@ohri.ca if you have any comments, corrections or questions.ogicinfo@ohri.ca See associated bibliography for references from this presentation and further reading. Thanks for your attention!

26. Matching genes to tags SAGEMap (NCBI) –From UniGene/ESTs 1,074,067 tags Build your own –from RefSeq 306,970 tags, 28,903 rank 0 tags –from Ensembl mRNA 322,076 tags, 34,050 rank 0 tags –from Ensembl genomic sequence 24,453,442 tags

27. Use of SAGE or Affymetrix

28. Tags in different databases Pou5f1 (Oct-4) –RefSeq 5 tags –SAGEMap 20 tags Nucleolin (Ncl) –RefSeq 9 tags –SAGEMap 223 tags

29. What do SAGE data look like?

30. How are SAGE data analysed

31. Computational generation of SAGE tag libraries From cDNA

32. What SAGE tag libraries are available

33. How can SAGE data be associated with Affy data

34. SAGE libraries in StemBase

35. Associating SAGE with Affy in StemBase