Serial Analysis of Gene Expression
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1 Serial Analysis of Gene Expression Cloning of Tissue-Specific Genes Using SAGE and a Novel Computational Substraction Approach. Genomic (2001) Hung-Jui Shih
2 Outline of Presentation SAGE EST Article TPE algorithm Results & Conclusion Reference
3 Serial Analysis of Gene Expression (SAGE) Developed in 1995 (Velculescu et al. Science 270 (5235) : ) Allows the quantitative and simultaneous analysis of a large number of transcripts. Identify novel expressed genes.
4 Principles of SAGE 1. Short sequence tag contains sufficient information to uniquely identify a transcript
5 Principles of SAGE 2. Sequence tags can be linked together to form long serial molecules that can be cloned and sequenced.
6 Principles of SAGE 3. Quantization of the number of times a particular tag observed provides the expression level of the corresponding transcript.
7 Serial Analysis of Gene Expression (SAGE)
8 Serial Analysis of Gene Expression (SAGE)
9 Serial Analysis of Gene Expression (SAGE)
10 Function of SAGE Software Identifies enzyme sites with proper spacing Extracts tags Record tags in database Match tags to genome sequence
11 Advantages of SAGE Highly sensitive - scaleable. Detects all genes including unknowns - quantitative data. Avoids amplification bias. Immortalized data allows for multiple comparisons. Circumvented unwanted crosshybridization
12 New in SAGE LongSAGE Characterizes a 21-base pair segment Saha S. et al. Using the transcriptome to annotate the genome (2002). 20(5), SAGE database I-SAGE kit (Invitrogen )
13 Expressed Sequence Tag (EST) Developed in (Adams M. et al. Science 252: ) A small part of the active part of a gene, made from cdna, bps, a kind of STS (sequence tagged site). Can be used to fish the rest of the gene out of the chromosome, by matching base pairs with part of the gene. Can be radioactively labeled in order to locate it in a larger segment of DNA.
14 Expressed Sequence Tag (EST)
15 Practical Advantages of EST Sequences can be generated rapidly and inexpensively Only one sequencing experiment is needed per each cdna generated Do not have to be checked for sequencing errors as mistakes. Do not prevent identification of the gene from which the EST was derived.
16 Challenge of EST Sequence is with errors including base insertion and deletion. Genes with lower expression are not easier to be picked which may have significant functions. It can be solved by... Normalization With large amount of EST
17 Summary of Characteristics SAGE Highly sensitive, efficient, comparability, technically demanding and needs a sequenced genome EST Sequence rapidly, inexpensive and business valuable (ex: Merck-EST project, (1998) Bioinformatics 14(1):2-13 ). Microarray Large-Scale, sensitive, technically challenging, limited to known genes and expensive
18 Cloning of Tissue-Specific Genes Rational Defect in expressed genes results in clinical phenotypes. Objective Identify functionally specialized genes. Material & Method No-match tags SAGE EST TPE algorithm
19 Tissue Preferential Expression (TPE) Based on the number of tissues in which a tag is present (The range of expression) and its expression level in the tissue of interest compared with other tissues (The preferential abundance). Calculate and plot scores Achieved as the Euclidean distance.
20 The preferential abundance M: tag count in the tissue of interest N j: tag count in another tissue j Add to each tag count: prevent division by 0 or taking the logarithm of 0
21 Results
22 Results TPE levels >= 7 are considered indicative for tissue specificity.
23 Results
24 Results
25 Conclusion Computational substraction of SAGE tags by the proposed TPE algorithm is a rapid and reliable way to expedite the cloning of tissue-specific genes.
26 Reference Patino W. et al (2002) Serial Analysis of Gene Expression: Technical Consideration and Application to Cardiovascular Biology. Circ Res 91: Velculescu, Zhang, Vogelstein & Kinzler. (1995) Serial Analysis of Gene Expression. Science 270 (5235) : Adams M. et al (1991) Complementary DNA Sequencing: Expressed Sequence Tags and Human Genome Project. Science 252:
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