Applications
Expression tags
Overview
The Genome Sequencer FLX System is an ideal platform for efficient, high-throughput direct sequencing of ditags for Serial Analysis of Gene Expression Ditags (SAGE). SAGE allows an accurate quantification and quantitative analysis of cell transcription in a variety of physiological and pathological conditions. Obtain an unbiased, high-quality gene expression profile using SuperSAGE ditag sequencing (2x26 nucleotides), which requires read lengths in excess of 75 bases.
Application Note:
Publications:
- Eveland et al. Transcript Profiling by 3'UTR Sequencing Resolves Expression of Gene Families. Plant Physiology 146: 32-44. January 2008.
- Shin et al. Transcriptome analysis for Caenorhabditis elegans based on novel expressed sequence tags (ESTs). BMC Biology 6: 30. July 8, 2008.
- Toth et al. Wasp Gene Expression Supports an Evolutionary Link Between Maternal Behavior and Eusociality. Science 318: 441-4. October 19, 2007.
Click here to see all Expression Tag publications.
How it Works:
SAGE: See Application Note
The substrate for transcriptome analysis usually consists of low molecular weight cDNA molecules such as ditags or short PCR products with 3' and 5' sequence information of mRNAs. Two methods can be employed to add "A" and "B" adaptor sequences needed for the subsequent emulsion PCR and sequencing reaction:
- 1. Ligation of the 44-mer standard A and B adaptors to cDNA molecule
- 2. Incorporation of A and B adaptor sequences during cDNA generation using Fusion Primers as described for Amplicon Sequencing
Figure 1: Generation of paired-end tags (PETs) and diPETs serving as substrate for adaptor ligation, emulsion PCR and subsequent sequencing.
Simultaneous sequencing of 5´ and 3´ ends of full-length cDNA molecules - Genome Identification Signature Sequencing (GIS-PET)
The Genome Identification Signature (GIS) technique was developed by the Singapore Genome Center (see Figure 3). GIS is a DNA-tag sequencing and mapping strategy in which 5' and 3' signatures of full-length cDNAs are accurately extracted into paired-end ditags (PETs) that are concatenated for efficient sequencing. The PETs are subsequently mapped to genome sequences to demarcate the transcription boundaries of every gene. GIS analysis is potentially 30-fold more efficient than standard (Sanger) cDNA sequencing approaches for transcriptome characterization. Using this approach, it is also possible to uncover intergenically spliced and unusual fusion transcripts. Paired-end ditagging for transcriptome analysis can also be applied to whole genome analysis of cis-regulatory and other DNA elements and represents an important technological advance for genome annotation.
Figure 3: Genome Identification Signature Sequencing with paired-end ditags (GIS-PET).