Challenges and considerations for reproducibility of STARR-seq assays
- Maitreya Das1,2,3,
- Ayaan Hossain3,4,
- Deepro Banerjee3,4,
- Craig Alan Praul3 and
- Santhosh Girirajan1,2,3,4,5
- 1Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, Pennsylvania 16802, USA;
- 2Molecular and Cellular Integrative Biosciences Graduate Program, Pennsylvania State University, University Park, Pennsylvania 16802, USA;
- 3Huck Institutes of the Life Sciences, Pennsylvania State University, University Park, Pennsylvania 16802, USA;
- 4Bioinformatics and Genomics Graduate Program, Pennsylvania State University, University Park, Pennsylvania 16802, USA;
- 5Department of Anthropology, Pennsylvania State University, University Park, Pennsylvania 16802, USA
Abstract
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.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.277204.122.
- Received August 14, 2022.
- Accepted March 15, 2023.
This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see https://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.
