SAGE data in StemBase Christopher Porter Ottawa Health Research Institute.

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SAGE data in StemBase Christopher Porter Ottawa Health Research Institute

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

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

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

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

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

Finding tags for a gene >gi| |ref|NM_ | 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

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 |

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

Associating tags with probesets

UCSC Genome Browser controls

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

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

Use of SAGE or Affymetrix

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

What do SAGE data look like?

How are SAGE data analysed

Computational generation of SAGE tag libraries From cDNA

What SAGE tag libraries are available

How can SAGE data be associated with Affy data

SAGE libraries in StemBase

Associating SAGE with Affy in StemBase