Overview

The Digital Gene Expression Displayer analyzes the differences in gene expression between two pools of libraries. Unlike the cDNA xProfiler, which lists every gene (even if an EST is seen only once in a pool) in both groups, the DGED finds only the statistically significant differences, based on the sequence odds ratio and a Bayesian test .

What to Put in the Query Fields

The initial selection of library pools A and B is similar to the cDNA xProfiler.

Query Field	Options
1. Organism	Select "Homo sapiens" or "Mouse" from the drop down box.
2. Library Group	There are three options: Keep the default setting of "All EST Libraries" to search all cDNA libraries in dbEST. Select MGC libraries (The Mammalian Gene Collection) or CGAP libraries or ORESTES libraries. Select any grouping of MGC, CGAP, and ORESTES.
3. Minimum sequences	The minimum number of sequences per cDNA library is set to 1000 by default. However, a user may enter a lower minimum number to include libraries of interest with the understanding the significance of the results may be lowered.
4. List libraries by	Select any option to organize the list of libraries for review on the set-up page.
5. Pools A and B
i) Tissue Type	There are 4 options: The default setting is all tissues (nothing is highlighted) with the "Include" button selected. This searches all tissue types. Keep the default setting of all tissues, check "Exclude", and highlight one or more** tissues. This will search all the tissues in the list excluding the highlighted item(s). Select a single tissue and keep the "Include" button checked. Select two or more tissues and keep the "Include" button checked.
ii) Tissue Preparation	The default setting is all library preparation methods (described in Tissue Preparation Overview) or choose one specific method or several.
iii) Tissue Histology	The default setting is normal, pre-cancer, and cancer histology, or choose one specific histology or several.
iv) Library Protocol	The default setting is all library protocol methods, or choose one specific protocol or several. The protocols listed below the line are CGAP specific protocols described in cDNA Library Protocols Overview.
v) Library Name	Enter a full or partial library name, e.g. NCI_CGAP_Pr1, NIH_MGC_50, or Pr.

** To choose multiple items in a select box, hold down the following keys together as you click on each item:

• In a PC: [CTRL] and [Alt]
• In a MAC: [Alt] and [Apple]

Examples of DGED Queries

The range of possible queries is the same of for the cDNA xProfiler, with the additional inclusion of the SAGE libraries. Below, the xProfiler examples are repeated with additional queries.

Pool A	Pool B
Human, normal breast	Human breast cancer
Human brain cancer	Any human tissue, excluding brain
Human cell lines of prostate cancer	Human bulk prostate cancer
Normal human colon, normalized	Normal human colon, non-normalized
Mouse CGAP library NCI_CGAP_Mam6	Mouse CGAP mammary libraries

Reviewing Selected Libraries in Pools A and B

Having selected the criteria for Pools A and B and pressed Submit Query, the "Review of Library Pools for DGED" page appears, which contains:

• The option to specify the expression factor (F). The expression factor, in conjunction with the significance filter (P), determines which results are reported. A result is reported if the odds ratio is significantly greater than F or significantly less than 1/F. F is set by default to "2" but this number may be set to any number greater than or equal to 1. As F increases, fewer results will be reported.
• The option to specify the significance filter (P). The significance filter, in conjunction with the expression factor (F), determines which results are reported. A result is reported if its significance is less than P. P is set by default to ".01" but this number may be set to any number from 0 to 1. As P increases, more results will be reported.
• Libraries chosen for each pool listed in a table.
  • The first column is the Target Pool (A) and the second column is "Background (B). The checked boxes indicate which library belongs to which pool.
  • The next column provides the number of sequences in the library.
  • The last column lists keywords which describe the library.

  It may be necessary to resort the libraries, e.g., to have all of Target followed by all Background libraries. To do this, click the back button on your browser and choose the appropriate criteria in #4. Press submit again.

Carefully review the libraries before proceeding. Check there are no libraries of "pooled" tissues or whole fetal tissue which may invalidate the results. Check that a library is not in both groups. Remove certain libraries to narrow your original selection. When you are satisfied, press Submit Query.

Understanding the DGED Results Page

The results page contains:

• The UniGene Build number
• The total number of sequences or tags in each pool
• The total number of libraries in each pool
• A table listing the genes or tags found to be expressed with a statistically significant difference between pools A and B. The following information is provided:

Field	Description
Symbol	The gene symbol for the gene cluster that contains two or more found sequences from the target pool.
Gene Info	A link to the Gene Info page, which contains links to gene information in other NCBI and NCI databases.
Accession Number	Representative GenBank accession number(s) for the gene
Libs A (or B)	A link to the libraries which contain this gene
Seqs A (or B)	The number of sequences found for a gene in either Pool A or B.
Seq Odds (A:B)	The sequence to odds ratio uses a simple mathematical formula to provide a measure of the relative amount of a gene in pool A to Pool B.
P value	A test of probability: the smaller the number, the more likely the result is not due merely to sampling error

Used together, the seqs odds ratio and the significance test provide a measure of confidence that the difference in the expression of a gene or tag is "real" and not due sampling error.