Challenges and considerations for reproducibility of STARR-seq assays

Area of Science:

• Genomics and Molecular Biology
• Epigenetics and Gene Regulation

Background:

• High-throughput sequencing methods like RNA-seq, ChIP-seq, and ATAC-seq are standardized, facilitating genome function studies.
• STARR-seq (Self-Transcribing Activated Regulatory Region sequencing) quantifies thousands of enhancer activities simultaneously but lacks standardization.
• Assay length and protocol/bioinformatic variations hinder STARR-seq reproducibility.

Purpose of the Study:

• To identify critical steps and quality control (QC) checkpoints for reproducible STARR-seq experiments.
• To provide guidelines for experimental design, protocol optimization, and bioinformatics analysis.
• To enhance the adoption, comparison, and integration of STARR-seq data across studies.

Main Methods:

• Systematic assessment of each step in the STARR-seq protocol and associated analysis pipelines.
• Evaluation of published STARR-seq protocols and in-house experimental assays.
• Identification of critical quality control points throughout the workflow.

Main Results:

• Critical steps and essential QC checkpoints for STARR-seq reproducibility were identified.
• Guidelines for experimental design, protocol scaling, customization, and bioinformatics pipelines were developed.
• Resources are provided to optimize STARR-seq for specific research applications.

Conclusions:

• Standardization of STARR-seq protocols and analysis is crucial for reliable enhancer activity quantification.
• The developed guidelines will improve the consistency and comparability of STARR-seq results.
• Enhanced reproducibility will facilitate broader adoption and data integration in regulatory genomics research.