SAGE data in StemBase

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SAGE data in StemBase

• Christopher Porter
• Ottawa Health Research Institute

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Presentation outline

• SAGE protocol
• SAGE analysis
• Integration with Affymetrix data
• Access to SAGE data in StemBase

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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

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Sequence to Tags
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Library database
----------------------------- tagSeq
tagCount -----------------------------
CTCGAGTTTCTTCCTGT 58
GGACAGCCGGGGATGCT 1
GTGGCTCACAACCATCT 461
TGAAGAGAGATATATCA 3
TGACAGAGCCAGGGCTC 6
TGGGAAGTGTGATTTCT 92
TTAATATTTAATTAGAG 56
TTTTATTTATATTCAAG 2 ------------------
-----------
GTGGCTCACAACCATCT TGACAGAGCCAGGGCTC TTAATATTTAATTA
GAG TGGGAAGTGTGATTTCT TTTTATTTATATTCAAG GGACAGCCGG
GGATGCT CTCGAGTTTCTTCCTGT TGAAGAGAGATATATCA
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SAGE tag identification

• Match to tags predicted from known sequences
• e.g. SAGEMap
• Generate mappings from cDNA sequences

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Finding tags for a gene
gtgi7305398refNM_013633.1 Mus musculus POU
domain, class 5, transcription factor 1
(Pou5f1), mRNA GTGAGCCGTCTTTCCACCAGGCCCCCGGCTCGGGG
TGCCCACCTTCCCCATGGCTGGACACCTGGCTTCA GACTTCGCCTCCTC
ACCCCCACCAGGTGGGGGTGATGGGTCAGCAGGGCTGGAGCCGGGCTGGG
TGGATT CTCGAACCTGGCTAAGCTTCCAAGGGCCTCCAGGTGGGCCTGG
AATCGGACCAGGCTCAGAGGTATTGGG GATCTCCCCATGTCCGCCCGCA
TACGAGTTCTGCGGAGGGATGGCATACTGTGGACCTCAGGTTGGACTG G
GCCTAGTCCCCCAAGTTGGCGTGGAGACTTTGCAGCCTGAGGGCCAGGCA
GGAGCACGAGTGGAAAGCA ACTCAGAGGGAACCTCCTCTGAGCCCTGTG
CCGACCGCCCCAATGCCGTGAAGTTGGAGAAGGTGGAACC AACTCCCGA
GGAGTCCCAGGACATGAAAGCCCTGCAGAAGGAGCTAGAACAGTTTGCCA
AGCTGCTGAAG CAGAAGAGGATCACCTTGGGGTACACCCAGGCCGACGT
GGGGCTCACCCTGGGCGTTCTCTTTGGAAAGG TGTTCAGCCAGACCACC
ATCTGTCGCTTCGAGGCCTTGCAGCTCAGCCTTAAGAACATGTGTAAGCT
GCG GCCCCTGCTGGAGAAGTGGGTGGAGGAAGCCGACAACAATGAGAAC
CTTCAGGAGATATGCAAATCGGAG ACCCTGGTGCAGGCCCGGAAGAGAA
AGCGAACTAGCATTGAGAACCGTGTGAGGTGGAGTCTGGAGACCA TGTT
TCTGAAGTGCCCGAAGCCCTCCCTACAGCAGATCACTCACATCGCCAATC
AGCTTGGGCTAGAGAA GGATGTGGTTCGAGTATGGTTCTGTAACCGGCG
CCAGAAGGGCAAAAGATCAAGTATTGAGTATTCCCAA CGAGAAGAGTAT
GAGGCTACAGGACACCTTTCCCAGGGGGGGCTGTATCCTTTCCTCTGCCC
CCAGGTCC CCACTTTGGCACCCCAGGCTATGGAAGCCCCCACTTCACCA
CACTCTACTCAGTCCCTTTTCCTGAGGGC GAGGCCTTTCCCTCTGTTCC
CGTCACTGCTCTGGGCTCTCCCATGCATTCAAACTGAGGCACCAGCCCTC
CCTGGGGATGCTGTGAGCCAAGGCAAGGGAGGTAGACAAGAGAACCTGG
AGCTTTGGGGTTAAATTCTTT TACTGAGGAGGGATTAAAAGCACAACAG
GGGTGGGGGGTGGGATGGGGAAAGAAGCTCAGTGATGCTGTT GATCAGG
AGCCTGGCCTGTCTGTCACTCATCATTTTGTTCTTAAATAAAGACTGGAC
ACACAGT
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4
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2
1
0
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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
---------------------------------------------
--------------------------------------------------
---
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Finding tags in genomic sequence
gt1 dnachromosome chromosomeNCBIM361472955047
295911 GAAACTGGCTCAGTGTAGCCATGAAGTCCAGGCCACTAACC
T

• 24,837,922 tags generated
• Tags observed at 1 - 36,168 locations
• 96 of tags are from a single location

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Associating tags with probesets
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UCSC Genome Browser controls
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Conclusion

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

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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

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Use of SAGE or Affymetrix
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Tags in different databases

• Pou5f1 (Oct-4)
• RefSeq
• 5 tags
• SAGEMap
• 20 tags
• Nucleolin (Ncl)
• RefSeq
• 9 tags
• SAGEMap
• 223 tags

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What do SAGE data look like?
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How are SAGE data analysed
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Computational generation of SAGE tag libraries

• From cDNA

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What SAGE tag libraries are available
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How can SAGE data be associated with Affy data
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SAGE libraries in StemBase
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Associating SAGE with Affy in StemBase
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