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Transcriptome-wide analysis of differential expression in perturbation atlases

Nature Genetics volume 57, pages 1228–1237 (2025)Cite this article

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Abstract

Single-cell CRISPR screens such as Perturb-seq enable transcriptomic profiling of genetic perturbations at scale. However, the data produced by these screens are noisy, and many effects may go undetected. Here we introduce transcriptome-wide analysis of differential expression (TRADE)—a statistical model for the distribution of true differential expression effects that accounts for estimation error appropriately. TRADE estimates the ‘transcriptome-wide impact’, which quantifies the total effect of a perturbation across the transcriptome. Analyzing several large Perturb-seq datasets, we show that many transcriptional effects remain undetected in standard analyses but emerge in aggregate using TRADE. A typical gene perturbation affects an estimated 45 genes, whereas a typical essential gene affects over 500. We find moderate consistency of perturbation effects across cell types, identify perturbations where transcriptional responses vary qualitatively across dosage levels and clarify the relationship between genetic and transcriptomic correlations across neuropsychiatric disorders.

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Fig. 1: Transcriptome-wide analysis of differential expression.
Fig. 2: Transcriptome-wide analysis of genome-wide Perturb-seq.
Fig. 3: TRADE-derived enrichment estimates.
Fig. 4: Correlation of differential expression across cell types.
Fig. 5: Dose–response relationships.
Fig. 6: Transcriptomic correspondence of neuropsychiatric conditions.

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Data availability

Raw sequencing data are deposited on SRA under BioProject PRJNA1100571. Aligned sequencing data and processed single-cell populations are available on GEO at GSE264667. Perturb-seq data from Replogle et al.6 can be accessed at https://gwps.wi.mit.edu/ (raw data available at SRA under BioProject PRJNA831566). dTAG data from Naqvi et al.26 are available at Gene Expression Omnibus (GEO) under accession number GSE205904. dTAG data from Weber et al.27 are available at GEO under accession number GSE145016. Summary statistics from the PsychENCODE consortium are available at https://github.com/mgandal/Shared-molecular-neuropathology-across-major-psychiatric-disorders-parallels-polygenic-overlap; Raw data are all available at Synapse under accession number syn4921369. RNA-seq data from the OneK1K dataset are available at GEO under accession number GSE196830.

Code availability

The TRADE method, with accompanying documentation, is publicly available as an R package at https://github.com/ajaynadig/TRADEtools. The publication version of this package is available as a persistent repository via Zenodo at https://doi.org/10.5281/zenodo.14993815 (ref. 58).

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Acknowledgements

We thank K. A. Lagattuta, D. J. Weiner, B. Harris, T. Aicher, D. L. Barabasi, K. Maher, T. Kamath, M. T. Tegtmeyer and members of the O’Connor and Robinson laboratories for helpful comments and discussions. A.N. is supported by National Institutes of Health (NIH) grant no. F31HG013036. J.M.R. is supported by NIH grant nos. F31NS115380, T32GM007618. This work was supported by a grant from SFARI (704413, E.B.R.). This work was supported by the Stanley Center for Psychiatric Research at the Broad Institute. L.J.O. acknowledges funding from the NIH National Institute of General Medical Sciences, 1R35GM155278, and SFARI, GR0243225. J.S.W. acknowledges funding from the NIH Center of Excellences in Genome Sciences, 2RM1HG009490, the Whitehead Innovation Initiative and The Eric and Wendy Schmidt Center at the Broad Institute. The project described was supported by award no. T32GM007753 and T32GM144273 from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the NIH. J.S.W. and S.A.M. are HHMI investigators.

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Authors and Affiliations

  1. Department of Biomedical Informatics, Harvard Medical School, Boston, MA, USA

    Ajay Nadig & Luke J. O’Connor

  2. Analytic and Translational Genetics Unit, Massachusetts General Hospital, Boston, MA, USA

    Ajay Nadig & Elise B. Robinson

  3. Stanley Center for Psychiatric Research, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Ajay Nadig, Mukundh Murthy, Steven A. McCarroll & Elise B. Robinson

  4. Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA

    Ajay Nadig & Elise B. Robinson

  5. Department of Medicine, Massachusetts General Hospital, Boston, MA, USA

    Joseph M. Replogle

  6. Medical Scientist Training Program, University of California San Francisco, San Francisco, CA, USA

    Joseph M. Replogle

  7. Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Joseph M. Replogle, Angela N. Pogson & Jonathan S. Weissman

  8. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Steven A. McCarroll

  9. Howard Hughes Medical Institute, Cambridge, MA, USA

    Steven A. McCarroll & Jonathan S. Weissman

  10. David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jonathan S. Weissman

  11. Department of Biology, Massachusetts Institute of Technology, Cambridge, MA, USA

    Jonathan S. Weissman

  12. Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Luke J. O’Connor

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Contributions

A.N., E.B.R. and L.J.O. conceived the study. J.M.R. and A.N.P. collected new data. J.S.W. supervised new data collection. A.N., M.M. and L.J.O. conducted analyses. A.N., J.M.R., M.M. and L.J.O. wrote the manuscript. S.A.M. provided additional project supervision.

Corresponding authors

Correspondence to Ajay Nadig, Joseph M. Replogle or Luke J. O’Connor.

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Competing interests

J.S.W. declares outside interest in 5 AM Venture, Amgen, Chroma Medicine, KSQ Therapeutics, Maze Therapeutics, Tenaya Therapeutics, Tessera Therapeutics, Ziada Therapeutics and Third Rock Ventures. J.M.R. consults for Third Rock Ventures and Maze Therapeutics, and is a consultant for and equity holder in Waypoint Bio. The remaining authors declare no competing interests.

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Nadig, A., Replogle, J.M., Pogson, A.N. et al. Transcriptome-wide analysis of differential expression in perturbation atlases. Nat Genet 57, 1228–1237 (2025). https://doi.org/10.1038/s41588-025-02169-3

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