Processing steps
Import data, setting all negative values to 0.
On a plate by plate basis:
Calculate the average of all wells labeled “blank” to obtain plate specific backgound signal
Subract backgound from all signals to obtain background subtracted values (bkgrnd_sub below) which are used in all further calculations
Set all background subtracted values that are less than zero to zero
(4.5 For layouts utilizing duplicates (2S4T, 4S2T), average the duplicates)
- Calculate norm, norm_pos, p_enhance as described below
Background subtraction, normalization
Upon data import, raw values are stored and processed as described above, then the calculations below are performed to yield additional columns of stored data.
| column | Description |
|---|---|
| raw | imported raw data |
| bkgrnd_sub | mean of all wells annotated “blank” subtracted from each raw
value; |
| norm | all values normalized to the maximum of the background subtracted
values annotated as “unknown”; |
| norm_pos | all values normalized to the mean of the background subtracted
values annotated as “positive”; |
| p_enhance | Percent enhancement over the positive control; 100*( |
Hit identification
Algorithm
| Label | Hit threshold |
|---|---|
| mean(neg) + 3SD | |
| mean(neg) + 2SD | |
| >0% enhanced | |
| Top N | Highest N responses from unknowns |
### References
Sittampalam GS, Coussens NP, Brimacombe K, et al., editors. Assay Guidance Manual Internet. Bethesda (MD): Eli Lilly & Company and the National Center for Advancing Translational Sciences; 2004-.
Brian P. Kelley, 1 Mitchell R. Lunn, 1 David E. Root, Stephen P. Flaherty, Allison M. Martino, and Brent R. Stockwell; A Flexible Data Analysis Tool for Chemical Genetic Screens, Chemistry & Biology 11:1495–1503, November, 2004