GUItars: a GUI tool for analysis of high-throughput RNA interference screening data.

High-throughput RNA interference (RNAi) screening has become a widely used approach to elucidating gene functions. However, analysis and annotation of large data sets generated from these screens has been a challenge for researchers without a programming background. Over the years, numerous data analysis methods were produced for plate quality control and hit selection and implemented by a few open-access software packages. Recently, strictly standardized mean difference (SSMD) has become a widely used method for RNAi screening analysis mainly due to its better control of false negative and false positive rates and its ability to quantify RNAi effects with a statistical basis. We have developed GUItars to enable researchers without a programming background to use SSMD as both a plate quality and a hit selection metric to analyze large data sets.The software is accompanied by an intuitive graphical user interface for easy and rapid analysis workflow. SSMD analysis methods have been provided to the users along with traditionally-used z-score, normalized percent activity, and t-test methods for hit selection. GUItars is capable of analyzing large-scale data sets from screens with or without replicates. The software is designed to automatically generate and save numerous graphical outputs known to be among the most informative high-throughput data visualization tools capturing plate-wise and screen-wise performances. Graphical outputs are also written in HTML format for easy access, and a comprehensive summary of screening results is written into tab-delimited output files.With GUItars, we demonstrated robust SSMD-based analysis workflow on a 3840-gene small interfering RNA (siRNA) library and identified 200 siRNAs that increased and 150 siRNAs that decreased the assay activities with moderate to stronger effects. GUItars enables rapid analysis and illustration of data from large- or small-scale RNAi screens using SSMD and other traditional analysis methods. The software is freely available at http://sourceforge.net/projects/guitars/

General workflow of high-throughput data analysis with GUItars.

<p>General workflow of high-throughput data analysis with GUItars.</p

User input files required by GUItars.

<p>Three separate input files are required by GUItars: A “data file directory” containing individual files for each plate, an “annotation file” with first two columns containing RNAi source plate ID and assay plate well ID with a single header line, and a “plate ID file” with a single header line. An “annotation file” and a “plate ID” file are mandatory only if the “hit mapping to the RNAi annotation file” option is checked.</p

Graphical outputs demonstrated on a 12-plate siRNA screen analyzed with the robust SSMD method with GUItars.

<p>(A) Raw data (left) and log₂-transformed data (right) histograms of each plate showing the original data distribution and effect of data transformation (one representative plate is shown). (B) Original scale (left) and rescaled (right) heat maps of each plate helping to capture systematic errors (one representative plate is shown). (C) Column-wise plate-series plot. (D) Screen-wise line plot for average control readings showing a clear separation between negative control and positive controls that is consistent throughout the screen. (E) Screen-wise SSMD score scatter plots with cutoff lines at 1.28 and −1.28 for signal-increasing and signal-decreasing hits, respectively. (F) Hit distribution heat maps for signal-increasing (top) and signal-decreasing (bottom) hits. (G) Screen-wise hit counts for signal-increasing (top) and signal-decreasing (bottom) hits.</p

Excel readable output file.

<p>Individual tab-delimited output files as well as a comprehensive Excel file are generated with the following information: Plate QC calculations before and after control outlier knockout, scores for all wells classified by well type, scores for hit wells classified by hit type (i.e., signal-increasing or signal-decreasing), and annotated hit list (optional) with corresponding scores.</p

Four p27^Kip1 inhibitors and calculated IC₅₀ values that passed all primary and secondary screens.

<p>The activity data for individual compounds were fit into sigmoidal dose-response curves (n = 3 per compound, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091173#pone-0091173-g003" target="_blank">Fig. 3C–F</a>) to derive IC₅₀ values.</p