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Mitochondrial single-cell ATAC-seq for high-throughput multi-omic detection of mitochondrial genotypes and chromatin accessibility

Nature Protocols volume 18, pages 1416–1440 (2023)Cite this article

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Abstract

Natural sequence variation within mitochondrial DNA (mtDNA) contributes to human phenotypes and may serve as natural genetic markers in human cells for clonal and lineage tracing. We recently developed a single-cell multi-omic approach, called ‘mitochondrial single-cell assay for transposase-accessible chromatin with sequencing’ (mtscATAC-seq), enabling concomitant high-throughput mtDNA genotyping and accessible chromatin profiling. Specifically, our technique allows the mitochondrial genome-wide inference of mtDNA variant heteroplasmy along with information on cell state and accessible chromatin variation in individual cells. Leveraging somatic mtDNA mutations, our method further enables inference of clonal relationships among native ex vivo-derived human cells not amenable to genetic engineering-based clonal tracing approaches. Here, we provide a step-by-step protocol for the use of mtscATAC-seq, including various cell-processing and flow cytometry workflows, by using primary hematopoietic cells, subsequent single-cell genomic library preparation and sequencing that collectively take ~3–4 days to complete. We discuss experimental and computational data quality control metrics and considerations for the extension to other mammalian tissues. Overall, mtscATAC-seq provides a broadly applicable platform to map clonal relationships between cells in human tissues, investigate fundamental aspects of mitochondrial genetics and enable additional modes of multi-omic discovery.

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Fig. 1: Schematic of the mtscATAC-seq experimental workflow.
Fig. 2: Schematic of the computational mtscATAC-seq pipeline.
Fig. 3: Flow cytometry cell sorting strategies.
Fig. 4: Images of human PBMCs before and after lysis.
Fig. 5: mtscATAC-seq library size distribution of human blood and immune cells.
Fig. 6: Troubleshooting mtscATAC-seq library size distribution.
Fig. 7: Overview of mtscATAC-seq computational workflow and quality-control metrics.

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Data availability

Raw mtscATAC-seq data for the demonstration of the analysis was downloaded from https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM4472967 and processed with CellRanger v2 to the hg38 reference genome before being processed with mgatk v0.6.1 The comparison single-nucleus ATAC-seq dataset is available at https://www.10xgenomics.com/resources/datasets/5-k-peripheral-blood-mononuclear-cells-pbm-cs-from-a-healthy-donor-next-gem-v-1-1-1-1-standard-2-0-0.

Code availability

A code resource for performing mtscATAC-seq analyses and reproducing the analysis figures in this work is available at https://github.com/caleblareau/mtscatac_protocol. The mgatk package is made freely available and is actively maintained at https://github.com/caleblareau/mgatk. Additional support for interactive variant calling in the R environment has been incorporated in the CRAN Signac package for single-cell chromatin analyses49.

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Acknowledgements

We are grateful to members of the Satpathy, Regev, Sankaran and Ludwig laboratories for helpful discussions. This research was supported by a Stanford Science Fellowship (to C.A.L.) and a Parker Institute of Cancer Immunotherapy Scholarship (to C.A.L.), a Stanford Artificial Intelligence in Medicine and Imaging seed grant (to C.A.L.), a BroadIgnite Award (L.S.L.), R01 DK103794 (to V.G.S.), R33 HL120791 (to V.G.S.), a gift from the Lodish Family to Boston Children’s Hospital (to V.G.S.), the New York Stem Cell Foundation (NYSCF; to V.G.S.) and the Howard Hughes Medical Institute and Klarman Cell Observatory (to A.R.). V.G.S. is an NYSCF-Robertson Investigator. P.K. is an Associated Fellow of the Hector Fellow Academy. L.N. is funded by a fellowship from the MDC-NYU exchange program and is an Associated Fellow of the Hector Fellow Academy. L.S.L. is supported by the Berlin Institute of Health, an Emmy Noether fellowship by the German Research Foundation (DFG, LU 2336/2-1) and a Hector Research Career Development Award. A.T.S. is supported by the National Institutes of Health (NIH) U01CA260852, the Parker Institute for Cancer Immunotherapy, a Career Award for Medical Scientists from the Burroughs Wellcome Fund and a Pew-Stewart Scholars for Cancer Research Award. A.T.S., C.A.L. and L.S.L. are supported by NIH UM1HG012076.

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Authors and Affiliations

  1. Department of Pathology, Stanford University, Stanford, CA, USA

    Caleb A. Lareau, Vincent Liu, Katalin Sandor, Yajie Yin, Jacob C. Gutierrez & Ansuman T. Satpathy

  2. Parker Institute for Cancer Immunotherapy, San Francisco, CA, USA

    Caleb A. Lareau & Ansuman T. Satpathy

  3. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Christoph Muus & Vijay G. Sankaran

  4. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, MA, USA

    Christoph Muus

  5. Berlin Institute of Health at Charité–Universitätsmedizin Berlin, Berlin, Germany

    Samantha D. Praktiknjo, Lena Nitsch, Pauline Kautz & Leif S. Ludwig

  6. Max‐Delbrück‐Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin Institute for Medical Systems Biology (BIMSB), Berlin, Germany

    Samantha D. Praktiknjo, Lena Nitsch, Pauline Kautz & Leif S. Ludwig

  7. Department of Biology, Chemistry, Pharmacy, Freie Universität Berlin, Berlin, Germany

    Lena Nitsch

  8. Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany

    Pauline Kautz

  9. Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA, USA

    Karin Pelka & Ansuman T. Satpathy

  10. Genentech, South San Francisco, CA, USA

    Aviv Regev

  11. Division of Hematology/Oncology, Boston Children’s Hospital and Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA, USA

    Vijay G. Sankaran

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Contributions

C.A.L., L.S.L. and C.M. conceived and designed the protocol with supervision from A.R. and V.G.S. K.S. designed and optimized the solid tissue dissociation protocol. V.L. and J.C.G. refined the computational workflow with supervision from C.A.L. and A.T.S. L.N., P.K., K.S., Y.Y. and K.P. independently executed the protocol and provided critical feedback on the manuscript. C.A.L., S.D.P., A.T.S., A.R., V.G.S. and L.S.L. supervised various aspects of this study. All authors contributed to the writing and editing of the protocol.

Corresponding authors

Correspondence to Caleb A. Lareau, Aviv Regev, Vijay G. Sankaran or Leif S. Ludwig.

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Competing interests

The Broad Institute has filed a patent relating to the use of the technology described in this paper for which C.A.L., L.S.L., C.M., A.R. and V.G.S. are named as inventors (US Patent App. 17/251,451). A.R. is a founder and equity holder of Celsius Therapeutics, an equity holder in Immunitas Therapeutics and until 31 August 2020, was a scientific advisory board member of Syros Pharmaceuticals, Neogene Therapeutics, Asimov and Thermo Fisher Scientific. From 1 August 2020, A.R. has been an employee of Genentech. V.G.S. serves as an advisor to and/or has equity in Novartis, Forma, Cellarity, Ensoma and Polaris Partners. A.T.S. is a founder of Immunai and Cartography Biosciences and receives research funding from Merck Research Laboratories and Allogene Therapeutics.

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Key references using this protocol

Lareau, C. et al. Nat. Biotechnol. 39, 451–461 (2021): https://doi.org/10.1038/s41587-020-0645-6

Walker, M. et al. N. Engl. J. Med. 383, 1556–1563 (2020): https://doi.org/10.1056/NEJMoa2001265

Penter, L. et al. Cancer Discov. 11, 3048–3063 (2021): https://doi.org/10.1158/2159-8290.CD-21-0276

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Lareau, C.A., Liu, V., Muus, C. et al. Mitochondrial single-cell ATAC-seq for high-throughput multi-omic detection of mitochondrial genotypes and chromatin accessibility. Nat Protoc 18, 1416–1440 (2023). https://doi.org/10.1038/s41596-022-00795-3

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