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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Raw and processed data have been deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE182685). Single-cell gene expression matrices, mtDNA variant calls and GoT results are available at https://vangalenlab.bwh.harvard.edu/resources/maester-2021/.
maegatk is available at https://github.com/caleblareau/maegatk, and a table with functional annotation of all possible mtDNA variants is available at https://github.com/EDePasquale/Mitochondrial_variants. Computational analyses are described at https://github.com/petervangalen/MAESTER-2021.
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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.
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 Figs. 1–17 and associated legends.
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). https://doi.org/10.1038/s41587-022-01210-8