A day late, and an shRNA short – how to avoid a low-power RNAi screen
Use our software tool to estimate screening power, before screening!
What’s the Problem?
RNA interference (RNAi) screening allows researchers to perform large-scale, whole-genome loss-of-function experiments to determine gene functions. These screens can identify genes that play roles in biochemical pathways, differentiation, genetic diseases and functional phenotypes. However, assessing the performance of a pooled shRNA screen is difficult. One can estimate the performance only by using reproducibility as a proxy for power or by employing a large number of validated positive and negative controls, which can be time-consuming and costly.
Our Bioinformatics and R&D groups have developed and published an innovative solution – the Power Decoder simulator - to help. The Power Decoder simulator is an open-source software tool developed for generating shRNA pooled screening experiments in silico to estimate a screen’s statistical power before the experiment is performed. This tool can be used to simulate screens with various hit fold changes, PCR conditions, and shRNA fold coverages.
This work shows how the Power Decoder simulator can help scientists plan future screens and easily investigate the likely effects of various experimental factors in silico, saving both time and money.
Simulated data sets to estimate the power of a proposed screen
In this work, the simulated data sets, although not perfect models of the experimental ones, showed similar distributions to the actual data. This led to power estimates that were realistic upper limits on those calculated from laboratory experiments, thus validating their usefulness.
Factors affecting screening power
By extending the simulations to predict the power of a pooled shRNA screen at various numbers of biological replicates and sequencing coverages, it was shown that shRNA fold coverage is much more influential than the number of replicates in obtaining a high power. Further, power increases with sequencing coverage until plateauing at approximately 200 sequences per shRNA for even modest 1.5-fold change hits.
What about previous screens?
Data from existing screens can also be analyzed retrospectively to evaluate their power and thus estimate the completeness of their resulting hit lists.
In addition, the Power Decoder simulator can be used to streamline optimization of novel pooled screening technologies such as gene knockout screens employing the new clustered regularly interspaced short palindrome repeats (CRISPR)–associated nuclease Cas9.
The Power Decoder simulator enables...
The ability to do fast, easy, accurate power analyses before the screen is carried out. This will enable researchers to perform adequately powered experiments, and deliver reliable answers to crucial biological questions.
- Get the source code for the Power Decoder simulator
- View the scientific poster now! The Power Decoder simulator for the evaluation of pooled shRNA screen performance
- SMARTvector shRNA Lentiviral Platform: Elucidating the Role of Factors Important in Neurite Outgrowth - Application Note
- Decode lentiviral shRNA screening libraries offer an efficient and cost-effective method for screening large numbers of shRNAs without automation.