SAGE: Procedure

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SAGE Procedure
Digest with Anchoring enzyme NlaIII
http//www.sagenet.org/home/Description.htm
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Mapping SAGE tags to genes
AAAAAAA
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Tags
Position of NlaIII site
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Experimental SAGE tags
Predicted Gene Models
Genomic DNA Sequence
EST Data
No EST Data
UTR length distribution
Transcripts with EST support
Map tags
Transcripts without EST support
Digest in silico
Adjust 3 UTRs
Theoretical SAGE tags
Conceptual mRNAs
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Mapping SAGE tags to genes
AAAAAAA
AAAAAAA
AAAAAAA
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Internal polyA tracts
AAAAAAA
AAAAAAA
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NlaIII Partial Digestion
AAAAAAA
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Alternative Splicing
AAAAAAA
AAAAAAA
AAAAAAA
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Serial Analysis of Gene Expression

• How many genes can we identify with SAGE?
• How many have multiple tags?
• How many extra tags are due to internal polyAs?
• How many extra tags are due to NlaIII partial
  digestion
• Do genes with gt1 tag have higher expression
  levels?

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1. Extract theoretical SAGE tags from the virtual
   transcriptome -- make a note of which ones are
   upstream of a polyA tract
2. Identify which experimental SAGE tags could be
   due to partial digestion with NlaIII
3. Identify genes with multiple SAGE tags, subtract
   tags encountered in steps 1 and 2
4. Calculate the tag abundance for genes with just
   one tag and genes with multiple tags

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Starting material

1. The virtual transcriptome in a fasta file
2. A summary of tag abundance and tag-to-gene
   mapping from the website
3. A summary of tags that map to intron of known
   genes

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