Rank ordering plate data facilitates data visualization and normalization in high-throughput screening.

High-throughput screening (HTS) of chemical and microbial strain collections is an indispensable tool for modern chemical and systems biology; however, HTS data sets have inherent systematic and random error, which may lead to false-positive or false-negative results. Several methods of normalization of data exist; nevertheless, due to the limitations of each, no single method has been universally adopted. Here, we present a method of data visualization and normalization that is effective, intuitive, and easy to implement in a spreadsheet program. For each plate, the data are ordered by ascending values and a plot thereof yields a curve that is a signature of the plate data. Curve shape characteristics provide intuitive visualization of the frequency and strength of inhibitors, activators, and noise on the plate, allowing potentially problematic plates to be flagged. To reduce plate-to-plate variation, the data can be normalized by the mean of the middle 50% of ordered values, also called the interquartile mean (IQM) or the 50% trimmed mean of the plate. Positional effects due to bias in columns, rows, or wells can be corrected using the interquartile mean of each well position across all plates (IQMW) as a second level of normalization. We illustrate the utility of this method using data sets from biochemical and phenotypic screens.