**5. "Hit" selection methods**

in the decision process, especially for cell-based assays with inherently high signal variability. The power of the above mentioned parameters were discussed in multiple studies (Gribbon et al. 2005; Iversen et al. 2006; Macarron and Hertzberg 2009; Ste‐

3 x std(Cpos) + 3 x std(Sall)

**•** SSMD: It is an alternative quality metric to Z'- and Z-factor, which was recently devel‐ oped to assess the assay quality in HT screens (Zhang 2007a; Zhang 2007b). Due to its ba‐ sis on probabilistic and statistical theories, SSMD was shown to be a more meaningful parameter than previously mentioned methods for QC purposes. SSMD differs from Z' and Z-factor by its ability to handle controls with different effects, which enables the se‐ lection of multiple QC criteria for assays (Zhang et al. 2008a). The application of SSMDbased QC criterion was demonstrated in multiple studies in comparison to other commonly-used methods (Zhang 2008b; Zhang 2011b; Zhang et al. 2008a). Although SSMD was developed primarily for RNAi screens, it can also be used for small molecule

SSMD= mean(Cpos) <sup>+</sup> mean(Cneg)

mean(Cneg)

or

or

**Assay variability ratio (AVR)** 3 x std(Cpos) <sup>+</sup> 3 x std(Cneg)

**Z'-factor** 1 - 3 x std(Cpos) <sup>+</sup> 3 x std(Cneg)

**Z-factor** 1 - 3 x std(Cpos) <sup>+</sup> 3 x std(Sall)

**SSMD** mean(Cpos) - mean(Cneg)

**Table 2.** Summary of HT screening data QC methods.

mean(Cpos)- mean(Cneg) std(Cneg)

mean(Cpos)- mean(Cneg) std(Cpos)2+ std(Cneg)<sup>2</sup>






std(Cpos)2+ std(Cneg)<sup>2</sup>


std(Cpos)2+ std(Cneg)2 (19)

Z-factor=1 -

vens et al. 1998).

212 Drug Discovery

screens.

**Signal-to-noise (S/N)**

**Signal window (SW)**

**Signal-to-background (S/B)** mean(Cpos)

The main purpose of HT screens is to obtain a list of compounds or siRNAs with desirable activ‐ ity for further confirmation. Therefore, the ultimate goal of an HT screening campaign is to nar‐ row down a big and comprehensive compound or siRNA library to a manageable number of "hits" with low false discovery rates. While the initial library of test samples undergoes multi‐ ple phases of elimination, the most critical factor is to select as many true "hits" as possible. Af‐ ter data normalization is applied as necessary, "hit" selection is performed on the plates that pass the QC criterion. As stated previously in Section 2.1, HT processes in primary and confir‐ matory screens differ in design. The "hit" selection process following a primary screen is simi‐ lar for RNAi and small-molecule screens, where the screening run is often performed in single copy, and a single data point (obtained from either endpoint or kinetic reading) is collected for each sample. On the other hand, a confirmatory RNAi screen is typically performed in repli‐ cates using pooled or individual siRNA, while the confirmatory small-molecule screens are executed in dose-response mode. Here, we classify the "hit" selection methodologies in two major categories: primary and confirmatory screen analysis.
