|Q: How are filters used to search for genes that fit a particular expression profile?|
Filters are applied as layers. The first filter will select all genes from the entire database (i.e. all 13,639 transcripts in the An. gambiae genome) that meet the corresponding criteria. If a second filter is placed, all genes that meet the criteria from both the first filter as well as the second filter will be selected. There is no limit to how many filters may be applied.|
||Select genes that meet criteria based on differential gene expression between larvae, male, non-bloodfed female, and bloodfed female mosquitoes at different timepoints after bloodfeeding.|
||Select genes that are preferentially expressed in specific tissues in the mosquito: the fat bodies, midgut, and ovaries, according to expression at 24 hours after a bloodmeal.|
||Select genes that have been annotated with the corresponding keywords.|
||Select genes that contain specific transcription factor binding sites, DNA sequences within their control or coding regions, or specific length cDNAs.
|Q: How do I focus on a subset of genes that meet specific expression criteria?|
|A: Click on the "Filter database" link in the navigation menu. The following form will appear:|
||(a) Enter a numeric value for the fold differential you wish to filter for. For example, to search for genes expressed at a magnitude 10 times greater at stage X compared to stage Y, enter "10" in this field.|
||(b) Select whether you wish to search for up-regulation or down-regulation of gene expression between the two stages of interest (i.e. (c) and (d)).|
||(c and d) Select the two stages to apply the filter to. For example, to search for genes that are more highly expressed in non-bloodfed female mosquitoes than in male mosquitoes, choose the fold differential you wish to select for in (a), select up-regulation in (b), choose the male [M] stage in (c) and choose the non-bloodfed female [NBF] stage in (d).|
||(e) If you wish to simultaneously filter for genes that meet the stage expression criteria and also contain annotated keywords, enter the keywords you wish to search for now. Otherwise, leave this field blank.|
Biological objective: Isolate all genes with known protease activity that are 1) preferentially expressed in female mosquitoes, 2) up-regulated 10 fold three hours after bloodfeeding, and 3) primarily expressed in the mosquito midgut.
1. Sex-specificity: May be achieved by selecting genes preferentially expressed in female non-bloodfed females [NBF] when compared to male [M] mosquitoes. In this example, we will select genes that are expressed 2 fold higher in non-bloodfed female mosquitoes compared to male mosquitoes. As shown in the output below, 1763 genes meet this criterion.
2. Stage-specificity: To select genes that are up-regulated 10 fold three hours after bloodfeeding [BF 3h] compared to non-bloodfed mosquitoes [NBF], enter these parameters as a second expression filter to be applied on top of the 1763 gene matches found from the first filter, above. As shown in the output below, 36 out of the 1763 genes that met the first filter criterion meet this second criterion as well.
3. Function-specificity: A keyword filter can be used to find which of these 36 gene matches contain gene annotation indicating protease activity. This keyword filter could have been applied simultaneously with either of the first two filters applied above, but can also be applied as a separate filter, as shown below. Just enter the keyword in the appropriate field and leave all other field settings as they are.
4. Tissue-specificity: To select genes preferentially expressed in one tissue, expand the "Tissue filters" fields by clicking on the "tissue filters >>" link in the filter options menu, highlighted below. Enter the fold differential to filter for and select which tissues to compare. For this example, to find genes that are preferentially expressed in the midgut, the first tissue field will be "midgut", and the second tissue field will be "fat bodies" in one filter, and "ovaries" in a second filter, as shown below.
|Input (tissue filter 1 of 2, midgut expression greater than expression in fat bodies):|
|Output (4 gene matches):|
|Input (tissue filter 2 of 2, midgut expression greater than expression in ovaries):|
|Output (4 gene matches, i.e. all of the matches found in the previous filter match this second filter as well):|
|Gene expression profile for one of these matches (ENSANGT00000016509). Emphasis on preferential expression in midgut:|
Note: The above filters could have been applied in any order. Additionally, the keyword and tissue filters could have been applied simultaneously with the sex-specific and stage-specific expression filters if desired.