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Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations


The combination of single-cell transcriptomics with mitochondrial DNA variant detection can be used to establish lineage relationships in primary human cells, but current methods are not scalable to interrogate complex tissues. Here, we combine common 3′ single-cell RNA-sequencing protocols with mitochondrial transcriptome enrichment to increase coverage by more than 50-fold, enabling high-confidence mutation detection. The method successfully identifies skewed immune-cell expansions in primary human clonal hematopoiesis.

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Fig. 1: Targeted enrichment of mitochondrial transcripts enables discrimination between genetic clones.
Fig. 2: Genetic clones exhibit lineage bias in clonal hematopoiesis.

Data availability

Raw and processed data have been deposited in the Gene Expression Omnibus ( Single-cell gene expression matrices, mtDNA variant calls and GoT results are available at

Code availability

maegatk is available at, and a table with functional annotation of all possible mtDNA variants is available at Computational analyses are described at


  1. Giladi, A. & Amit, I. Single-cell genomics: a stepping stone for future immunology discoveries. Cell 172, 14–21 (2018).

    Article  CAS  Google Scholar 

  2. Acosta, J., Ssozi, D. & van Galen, P. Single-cell RNA sequencing to disentangle the blood system. Arterioscler. Thromb. Vasc. Biol. 41, 1012–1018 (2021).

    Article  CAS  Google Scholar 

  3. Nam, A. S. et al. Somatic mutations and cell identity linked by genotyping of transcriptomes. Nature 571, 355–360 (2019).

    Article  CAS  Google Scholar 

  4. van Galen, P. et al. Single-cell RNA-seq reveals AML hierarchies relevant to disease progression and immunity. Cell 176, 1265–1281 (2019).

    Article  CAS  Google Scholar 

  5. Wagner, D. E. & Klein, A. M. Lineage tracing meets single-cell omics: opportunities and challenges. Nat. Rev. Genet. 21, 410–427 (2020).

    Article  CAS  Google Scholar 

  6. Liggett, L. A. & Sankaran, V. G. Unraveling hematopoiesis through the lens of genomics. Cell 182, 1384–1400 (2020).

    Article  CAS  Google Scholar 

  7. Ludwig, L. S. et al. Lineage tracing in humans enabled by mitochondrial mutations and single-cell genomics. Cell 176, 1325–1339 (2019).

    Article  CAS  Google Scholar 

  8. Lareau, C. A. et al. Massively parallel single-cell mitochondrial DNA genotyping and chromatin profiling. Nat. Biotechnol. 39, 451–461 (2021).

    Article  CAS  Google Scholar 

  9. Xu, J. et al. Single-cell lineage tracing by endogenous mutations enriched in transposase accessible mitochondrial DNA. eLife 8, e45105 (2019).

    Article  CAS  Google Scholar 

  10. Velten, L. et al. Identification of leukemic and pre-leukemic stem cells by clonal tracking from single-cell transcriptomics. Nat. Commun. 12, 1366 (2021).

    Article  CAS  Google Scholar 

  11. Hughes, T. K. et al. Second-strand synthesis-based massively parallel scRNA-seq reveals cellular states and molecular features of human inflammatory skin pathologies. Immunity 53, 878–894 (2020).

    Article  CAS  Google Scholar 

  12. Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015).

    Article  CAS  Google Scholar 

  13. Mimitou, E. P. et al. Scalable, multimodal profiling of chromatin accessibility, gene expression and protein levels in single cells. Nat. Biotechnol. 39, 1246–1258 (2021).

    Article  CAS  Google Scholar 

  14. Tu, A. A. et al. TCR sequencing paired with massively parallel 3′ RNA-seq reveals clonotypic T cell signatures. Nat. Immunol. 20, 1692–1699 (2019).

    Article  CAS  Google Scholar 

  15. Abdel-Wahab, O. et al. Genetic characterization of TET1, TET2, and TET3 alterations in myeloid malignancies. Blood 114, 144–147 (2009).

    Article  CAS  Google Scholar 

  16. Street, K. et al. Slingshot: cell lineage and pseudotime inference for single-cell transcriptomics. BMC Genomics 19, 477 (2018).

    Article  Google Scholar 

  17. Moran-Crusio, K. et al. Tet2 loss leads to increased hematopoietic stem cell self-renewal and myeloid transformation. Cancer Cell 20, 11–24 (2011).

    Article  CAS  Google Scholar 

  18. Luchman, H. A. et al. An in vivo patient-derived model of endogenous IDH1-mutant glioma. Neuro. Oncol. 14, 184–191 (2012).

    Article  CAS  Google Scholar 

  19. Flavahan, W. A. et al. Insulator dysfunction and oncogene activation in IDH mutant gliomas. Nature 529, 110–114 (2016).

    Article  CAS  Google Scholar 

  20. Buenrostro, J. D., Giresi, P. G., Zaba, L. C., Chang, H. Y. & Greenleaf, W. J. Transposition of native chromatin for fast and sensitive epigenomic profiling of open chromatin, DNA-binding proteins, and nucleosome position. Nat. Methods 10, 1213–1218 (2013).

    Article  CAS  Google Scholar 

  21. Morgan, M., Obenchain, V., Hester, J. & Pagès, H. SummarizedExperiment: SummarizedExperiment container. R package version 1.24.0 (2019).

  22. Stuart, T. et al. Comprehensive integration of single-cell data. Cell 177, 1888–1902.e21 (2019).

    Article  CAS  Google Scholar 

  23. Yang, S. et al. Decontamination of ambient RNA in single-cell RNA-seq with DecontX. Genome Biol. 21, 57 (2020).

    Article  Google Scholar 

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We thank patients for donating cells; C. Couturier and M. Villanueva from the A. Shalek lab for sequencing; A. Kreso, V. Hovestadt and A. A. Tu for helpful discussions; and P. Rogers for technical support. P.v.G., A.A.L. and B.E.B. are supported by the Ludwig Center at Harvard. P.v.G. and V.G.S. are supported by the Harvard Medical School Epigenetics & Gene Dynamics Initiative. P.v.G. is supported by the National Institutes of Health (NIH) R00 Award (CA218832), Gilead Sciences, the Bertarelli Rare Cancers Fund, and is an awardee of the Glenn Foundation for Medical Research and American Federation for Aging Research (AFAR) Grant for Junior Faculty. T.E.M. is supported by the American Brain Tumor Association Basic Research Fellowship in honor of Joel A. Gingras, Jr. T.E.M. and J.A.V. are supported by the UK Brain Tumour Charities Future Leaders Award (GN-000701). C.A.L. is supported by a Stanford Science Fellowship and Parker Scholar award. A.T.S. is supported by NIH grant U01CA260852, the Cancer Research Institute Technology Impact Award and a Pew-Stewart Scholars for Cancer Research Award. L.S.L. is supported by an Emmy Noether fellowship by the German Research Foundation (LU 2336/2-1). J.C.L and D.M.M were supported in part by the Koch Institute Support (core) NIH grant P30-CA14051 from the National Cancer Institute, as well as the Koch Institute–Dana-Farber/Harvard Cancer Center Bridge Project and the Food Allergy Science Initiative at the Broad Institute. V.G.S. is supported by the New York Stem Cell Foundation, a gift from the Lodish family to Boston Children’s Hospital and NIH grant R01 DK103794. V.G.S. is a New York Stem Cell Foundation–Robertson Investigator.

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Authors and Affiliations



T.E.M., C.A.L., J.A.V., E.A.K.D., V.L., D.S., K.S., Y.Y., C.A.E.F., D.M.M., A.T.S. and P.v.G. conducted experiments and analyzed the data. T.E.M., C.A.L., L.S.L., G.K.G., A.A.L., J.C.L., B.E.B., V.G.S. and P.v.G. designed the study and interpreted the data. T.E.M. and P.v.G. wrote the manuscript. All authors edited the manuscript.

Corresponding author

Correspondence to Peter van Galen.

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Competing interests

B.E.B. discloses financial interests in Fulcrum Therapeutics, HiFiBio, Arsenal Biosciences, Chroma Medicine and Cell Signaling Technologies. V.G.S. serves as an advisor to and/or has equity in Novartis, Forma, Cellarity, Ensoma and Branch Biosciences. T.E.M. discloses financial interests in Telomere Diagnostics and Reify Health. A.T.S. discloses financial interests in Immunai and Cartography Biosciences. J.C.L. has interests in Honeycomb Biotechnologies. J.C.L.’s interests are reviewed and managed under the Massachusetts Institute of Technology’s policies for potential conflicts of interest. J.C.L. and the Massachusetts Institute of Technology have filed patents related to the single-cell sequencing methods used in this work. A patent application covering MAESTER has been filed by the Broad Institute of MIT and Harvard. The remaining authors declare no competing interests.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–17 and associated legends.

Reporting Summary.

Supplementary Table 1

All variants that were used to inform clonal structures, along with functional annotations. This is an excerpt from a table with annotations for every possible mitochondrial variant (Methods).

Supplementary Table 2

All oligo sequences used for MAESTER on Seq-Well scRNA-seq cDNA.

Supplementary Table 3

All oligo sequences used for MAESTER and TREK-seq on 10x Genomics 3′ v3 scRNA-seq cDNA.

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Miller, T.E., Lareau, C.A., Verga, J.A. et al. Mitochondrial variant enrichment from high-throughput single-cell RNA sequencing resolves clonal populations. Nat Biotechnol 40, 1030–1034 (2022).

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