Factors affecting reproducibility between genome-scale siRNA-based screens

Area of Science:

• Genomics
• Virology
• Bioinformatics

Background:

• RNA interference (RNAi) screening is a key genomic technology for understanding gene function.
• Whole-genome RNAi screens are powerful tools for identifying host factors in viral infections.

Purpose of the Study:

• To evaluate factors influencing hit list composition and reproducibility in RNAi screening.
• To compare different analysis methodologies for whole-genome RNAi screens.
• To assess the reproducibility of siRNA screening over time.

Main Methods:

• Performed two identical whole-genome small interfering RNA (siRNA)-based screens for yellow fever virus host factors.
• Compared candidate hit lists generated by various statistical methods (sum rank, median absolute deviation, z-score, strictly standardized mean difference).
• Assessed intra- and inter-screen reproducibility using different analysis approaches.

Main Results:

• Analysis methodology significantly impacted hit list composition, with results ranging from 82 to 1140 members.
• Reproducibility varied widely (32% to 99%) depending on the analysis method used.
• Testing at least two independent siRNAs per gene product in primary screens enhances validation.

Conclusions:

• The choice of analysis methodology is critical and profoundly influences RNAi screening outcomes.
• Standardized data sharing of functional genome-scale screening data is crucial for validation and reproducibility.
• Methods to reduce false discovery at the primary screening stage are essential.