Target discovery screens using pooled shRNA libraries and NGS: A model workflow and analytical algorithm

In the search for novel therapeutic targets, RNA interference screening has become a valuable tool. High-throughput technologies are now broadly accessible but their assay development from baseline remains resource-intensive and challenging. Focusing on this assay development process, researchers from the University Hospital Münster describe a target discovery screen using pooled shRNA libraries and next-generation sequencing (NGS) deconvolution in a cell line model of Ewing sarcoma. In a strategy designed for comparative and synthetic lethal studies, they screened for targets specific to the A673 Ewing sarcoma cell line. Methods, results and pitfalls are described for the entire multi-step screening procedure, from lentiviral shRNA delivery to bioinformatics analysis, illustrating a complete model workflow. The researchers demonstrate that successful studies are feasible from the first assay performance and independent of specialized screening units. Furthermore, they show that a resource-saving screen depth of 100-fold average shRNA representation can suffice to generate reproducible target hits despite heterogeneity in the derived datasets. Because statistical analysis methods are debatable for such datasets, they created ProFED, an analysis package designed to facilitate descriptive data analysis and hit calling using an aim-oriented profile filtering approach. In its versatile design, this open-source online tool provides fast and easy analysis of shRNA and other count-based datasets to complement other analytical algorithms.

General design and workflow of the pooled shRNA screen


Collected samples of representative cell populations are depicted in blue: ctrl_a/b = unselected control populations; exp_a/b = experimental populations selected for phenotype of interest, here cell survival; Decode ctrl = virus particle control.

Availability – the ProFED application is available at: or at:

Schaefer C, Mallela N, Seggewiß J, Lechtape B, Omran H, Dirksen U, et al. (2018) Target discovery screens using pooled shRNA libraries and next-generation sequencing: A model workflow and analytical algorithm. PLoS ONE 13(1): e0191570. [article]

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