Challenges and considerations for reproducibility of STARR-seq assays

Maitreya Das, Ayaan Hossain, Deepro Banerjee, Craig Alan Praul, Santhosh Girirajan

Research output: Contribution to journalArticlepeer-review


High-throughput methods such as RNA-seq, ChIP-seq, and ATAC-seq have well-established guidelines, commercial kits, and analysis pipelines that enable consistency and wider adoption for understanding genome function and regulation. STARR-seq, a popular assay for directly quantifying the activities of thousands of enhancer sequences simultaneously, has seen limited standardization across studies. The assay is long, with more than 250 steps, and frequent customization of the protocol and variations in bioinformatics methods raise concerns for reproducibility of STARR-seq studies. Here, we assess each step of the protocol and analysis pipelines from published sources and in-house assays, and identify critical steps and quality control (QC) checkpoints necessary for reproducibility of the assay. We also provide guidelines for experimental design, protocol scaling, customization, and analysis pipelines for better adoption of the assay. These resources will allow better optimization of STARR-seq for specific research needs, enable comparisons and integration across studies, and improve the reproducibility of results.

Original languageEnglish (US)
Pages (from-to)479-495
Number of pages17
JournalGenome research
Issue number4
StatePublished - Apr 2023

All Science Journal Classification (ASJC) codes

  • Genetics
  • Genetics(clinical)


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