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EST EXPRESSION PROFILE ANALYSIS??? ????

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EST

• Expressed Sequence Tags

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EST

• Expressed Sequence Tags are partial, single-pass
  sequences from either end of a cDNA clone.

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• Expressed Sequence Tag (EST) were generated by
  automated partial DNA sequencing of randomly
  selected cDNA clones from individual cDNA
  libraries.

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• EST sequences are real experimental data with
  about 1 per base error rate.

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Statistics

• dbEST

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Cancer Genome Anatomy Project(CGAP)

• Tissue
• Normal
• Precancer
• Cancer

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Cancer Genome Anatomy Project(CGAP)

• Tissue preparation
• Bulk
• Microdissected
• Cell lines
• Flow sorted

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Cancer Genome Anatomy Project(CGAP)

• cDNA library protocols
• Non-normalized
• Normalized
• Subtraction
• (In "native" non-normalized libraries, clone
  frequency reflects mRNA abundance)

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Cancer Genome Anatomy Project(CGAP)

• cDNA library protocols

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Cancer Genome Anatomy Project(CGAP)

• cDNA libraries
• Library finder
• Library list
• Library browser
• cDNA Library navigator
• Unified library database
• Example
• Summary

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EST cDNA library projects

• CGAP
• MGC
• ORESTES
• And others

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Serial Analysis of Gene Expression(SAGE)

• Diagram 1
• Diagram 2

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Serial Analysis of Gene Expression(SAGE)

• Restriction enzymes
• SAGE vs. EST
• Shorter tags (913 bp)
• Same sequencing power, more information
• More sensitive to sequencing error
• Tag-gene mapping is much messier

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Unigene

• Automatic clustering of ESTs
• Unique set of human and mouse genes
• Unigene and genome mapping
• Diagram

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Gene Index

• TGI TIGR Gene Indices
• EGI EuroGeneIndexes
• MIPS
• STACK

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EST as tools (1)

• Discover new genes
• Genome mapping
• cDNA assembly
• Cluster only Unigene
• Cluster assemble (alignment consensus)
• TGI EGI MIPS STACK
• Assemble curate
• EGAD

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EST as tools (2)

• Alternative splicing
• Expression profile comparison
• Digital differential display
• Xprofiler
• Digital Gene Expression displayer (DGED)
• Virtual northern

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Expression Profiles

• Protein profiles
• mRNA profiles

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How to obtain expression profiles

• Protein
• Proteomics
• Protien chips
• mRNA
• EST cDNA libraries
• SAGE
• Microarray

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Tools to use mRNA profiles

• Digital differential display
• Xprofiler
• Digital Gene Expression Displayer (DGED)
• Virtual northern
• BodyMap

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Our tools

• NCKU Xcrunch
• (eXpression profile CRUNCHer)
• Digital differential display
• Digital FISH
• Digital Northern

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Our database

• Automatically download the expression profiles
  from Unigene Library Browser
• Summary

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Digital differential display
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Digital FISH (1)

• Goal To narrow down genes responsible for our
  favorite cancers in certain chromosomal regions. 
  (For example renal cancer chromosome 3p22-23)

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Digital FISH (2)

• Rationale Genes related to kidney tumors should
  be expressed in kidney tissue (normal and/or
  abnormal), i.e., should be found in kidney
  related EST cDNA libraries.  (exceptions viral
  gene products Hormone-like effect)

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Digital FISH (3)

• Step 1 Get the Unigenes located in the
  chromosomal regions.
• Step 2 Get the Unigenes from kidney-related cDNA
  libraries.
• Step 3 What Unigenes are present in both 1 and 2?

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Digital Northern
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www.binfo.ncku.edu.tw/xcrunch/

• Database
• Xcrunch
• Some explanation

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Acknowledgement

• ??? (the programmer of NCKU Xcrunch)
• ?? ??

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The Mammalian Gene Collection(MGC)

• NIH
• To identify and sequence a representative full
  open reading frame (ORF) clone for each human and
  mouse gene

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The Mammalian Gene Collection(MGC)

• Libraries with up to at least 3 to 4 kb cDNA
  inserts
• EST sequences (5 3 Accuracy same as ESTs)
• Full-length candidates submitted for full insert
  sequencing (at least 99.99 accuracy Same as
  HGP)
• Back

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ORF expressed sequence tags(ORESTES)

• Reverse transcription-PCR using arbitrarily
  selected, nondegenerae primers under
  low-stringency
• High percentage of fragments in the midportions
  of genes
• Normalization of the sequence population

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From Neto et al. Shotgun sequencing of the human
transcriptome with ORF expressed sequence tags.
2000. PNAS 97(7) p3491-3496.
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Full length cDNA projects

• MGC
• FLJ
• RIKEN
• DKFZ

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Number of cDNA libraries
Homo sapiens 6908
Mus musculus 557
Rattus norvegicus 237
Xenopus laevis 49
Danio rerio (zebrafish) 49
Arabidopsis thaliana 66
BosTaurus 60
HordeumVulgare 44
OryzaSativa 69
TriticumAestivum 50
ZeaMays 44
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