1 / 31

Ligate tags

Isolate mRNA, RT to cDNA. Digest with “Tagging enzyme” Bsm FI. Sequence. Ligate tags. SAGE: Procedure. Digest with “Anchoring enzyme” Nla III. http://www.sagenet.org/home/Description.htm. Mapping SAGE tags to genes. AAAAAAA. Tags. Position of NlaIII site. Predicted Gene Models.

dalton-nielsen
Download Presentation

Ligate tags

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Isolate mRNA, RT to cDNA Digest with “Tagging enzyme” BsmFI Sequence Ligate tags SAGE:Procedure Digest with “Anchoring enzyme” NlaIII http://www.sagenet.org/home/Description.htm

  2. Mapping SAGE tags to genes AAAAAAA

  3. Tags Position of NlaIII site

  4. Predicted Gene Models Genomic DNA Sequence Experimental SAGE tags 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

  5. Mapping SAGE tags to genes AAAAAAA AAAAAAA AAAAAAA

  6. Internal polyA tracts AAAAAAA AAAAAAA

  7. NlaIII NlaIII NlaIII Partial Digestion AAAAAAA

  8. Alternative Splicing AAAAAAA AAAAAAA AAAAAAA

  9. SerialAnalysisofGeneExpression • 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 >1 tag have higher expression levels?

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

  11. Starting material: The virtual transcriptome in a fasta file A summary of tag abundance and tag-to-gene mapping from the website A summary of tags that map to intron of known genes

More Related