Challenges and considerations for reproducibility of STARR-seq assays

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 activity of thousands of enhancer sequences simultaneously, has seen limited standardization across studies. Further, the assay is long with >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 checkpoints necessary for reproducibility of the assay. We also provide guidelines for assay 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 reproducibility of results.

HDI-STARR-seq: Condition-specific enhancer discovery in mouse liver in vivo

“…Other limitations, which apply to STARR-seq in general, include the need for extensive PCR amplification when preparing high complexity STARR-seq reporter RNA libraries for sequencing, which tends to decrease library complexity and introduce variability in the recovery of individual reporters [87]. This issue can be addressed by inclusion of UMIs to eliminate PCR amplification bias during preparation of STARR-seq reporter sequencing libraries [60].…”

“…Promoter selection is a key parameter that impacts the level of reporter transcription in MPRAs, including STARR-seq. Different core promoters have preferences for different cofactors [28, 93] and genomic environments [94] and for enhancers targeting genes of particular function [87]. Overall, more than 50% of enhancers tested show significant preferences for specific promoters [95].…”

“…Introduction of naked plasmid DNA in STARR-seq-based transfection experiments elicits a strong type-I interferon innate immune response [25]. This response, which can also occur when plasmid DNA is transfected into mouse liver [86], is a serious confounding factor for STARR-seq, as it produces large numbers of enhancer false positives and false negatives [25], raising concerns about the reliability of enhancer activities determined by STARR-seq, even when kinase inhibitors are added to block type-I interferon induction [87]. In contrast, using HDI-STARR-seq, we found little or no sustained type-I interferon response when naked STARR-seq plasmid DNA was transfected into mouse liver, as determined both 20 h and 7 d after HDI.…”

High-resolution dissection of human cell type-specific enhancers incisandtransactivities

“…Thus, by high-throughput sequencing of the reporter RNA and mapping the test sequence back to genome, it can not only identify the genomic location of enhancer sequences, but also provide a quantitative map of enhancer activities across the genome. STARR-seq has been widely employed in genome-wide quantification of enhancer activities in different species and different types of cells (Das et al 2023). When applied to human and other mammalian cells (Liu et al 2017;Johnson et al 2018), due to the large size of their genomes, usually large fragment sizes have to be adopted if the whole genome is used as input, resulting in low-resolution enhancer sequences identified.…”