Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Sequencing thousands of single-cell genomes with combinatorial indexing

Nature Methods volume 14, pages 302–308 (2017)Cite this article

Subjects

Abstract

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Single-cell combinatorial indexing with nucleosome depletion.
Figure 2: Comparison of LAND and xSDS nucleosome depletion methods with SCI-seq.
Figure 3: Somatic CNVs in the rhesus brain.
Figure 4: SCI-seq analysis of a stage III human pancreatic ductal adenocarcinoma (PDAC).

Similar content being viewed by others

Accession codes

Primary accessions

Sequence Read Archive

References

  1. McConnell, M.J. et al. Mosaic copy number variation in human neurons. Science 342, 632–637 (2013).

    Article  CAS  Google Scholar 

  2. Cai, X. et al. Single-cell, genome-wide sequencing identifies clonal somatic copy-number variation in the human brain. Cell Rep. 8, 1280–1289 (2014).

    Article  CAS  Google Scholar 

  3. Knouse, K.A., Wu, J., Whittaker, C.A. & Amon, A. Single cell sequencing reveals low levels of aneuploidy across mammalian tissues. Proc. Natl. Acad. Sci. USA 111, 13409–13414 (2014).

    Article  CAS  Google Scholar 

  4. Rehen, S.K. et al. Chromosomal variation in neurons of the developing and adult mammalian nervous system. Proc. Natl. Acad. Sci. USA 98, 13361–13366 (2001).

    Article  CAS  Google Scholar 

  5. Navin, N. et al. Tumour evolution inferred by single-cell sequencing. Nature 472, 90–94 (2011).

    Article  CAS  Google Scholar 

  6. Eirew, P. et al. Dynamics of genomic clones in breast cancer patient xenografts at single-cell resolution. Nature 518, 422–426 (2015).

    Article  CAS  Google Scholar 

  7. Gawad, C., Koh, W. & Quake, S.R. Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics. Proc. Natl. Acad. Sci. USA 111, 17947–17952 (2014).

    Article  CAS  Google Scholar 

  8. Gao, R. et al. Punctuated copy number evolution and clonal stasis in triple-negative breast cancer. Nat. Genet. 48, 1119–1130 (2016).

    Article  CAS  Google Scholar 

  9. Zong, C., Lu, S., Chapman, A.R. & Xie, X.S. Genome-wide detection of single-nucleotide and copy-number variations of a single human cell. Science 338, 1622–1626 (2012).

    Article  CAS  Google Scholar 

  10. Baslan, T. et al. Optimizing sparse sequencing of single cells for highly multiplex copy number profiling. Genome Res. 25, 714–724 (2015).

    Article  CAS  Google Scholar 

  11. Knouse, K.A., Wu, J. & Amon, A. Assessment of megabase-scale somatic copy number variation using single-cell sequencing. Genome Res. 26, 376–384 (2016).

    Article  CAS  Google Scholar 

  12. Gawad, C., Koh, W. & Quake, S.R. Single-cell genome sequencing: current state of the science. Nat. Rev. Genet. 17, 175–188 (2016).

    Article  CAS  Google Scholar 

  13. Adey, A. et al. Rapid, low-input, low-bias construction of shotgun fragment libraries by high-density in vitro transposition. Genome Biol. 11, R119 (2010).

    Article  CAS  Google Scholar 

  14. Amini, S. et al. Haplotype-resolved whole-genome sequencing by contiguity-preserving transposition and combinatorial indexing. Nat. Genet. 46, 1343–1349 (2014).

    Article  CAS  Google Scholar 

  15. Adey, A. et al. In vitro, long-range sequence information for de novo genome assembly via transposase contiguity. Genome Res. 24, 2041–2049 (2014).

    Article  CAS  Google Scholar 

  16. 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 

  17. Cusanovich, D.A. et al. Multiplex single cell profiling of chromatin accessibility by combinatorial cellular indexing. Science 348, 910–914 (2015).

    Article  CAS  Google Scholar 

  18. Stergachis, A.B. et al. Developmental fate and cellular maturity encoded in human regulatory DNA landscapes. Cell 154, 888–903 (2013).

    Article  CAS  Google Scholar 

  19. ENCODE Project Consortium. An integrated encyclopedia of DNA elements in the human genome. Nature 489, 57–74 (2012).

  20. Adey, A. et al. The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line. Nature 500, 207–211 (2013).

    Article  CAS  Google Scholar 

  21. 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 

  22. Garvin, T. et al. Interactive analysis and quality assessment of single-cell copy-number variations. Nat. Methods 12, 1058–1060 (2015).

    Article  CAS  Google Scholar 

  23. Goryshin, I.Y., Miller, J.A., Kil, Y.V., Lanzov, V.A. & Reznikoff, W.S. Tn5/IS50 target recognition. Proc. Natl. Acad. Sci. USA 95, 10716–10721 (1998).

    Article  CAS  Google Scholar 

  24. Olshen, A.B., Venkatraman, E.S., Lucito, R. & Wigler, M. Circular binary segmentation for the analysis of array-based DNA copy number data. Biostatistics 5, 557–572 (2004).

    Article  Google Scholar 

  25. Ha, G. et al. Integrative analysis of genome-wide loss of heterozygosity and monoallelic expression at nucleotide resolution reveals disrupted pathways in triple-negative breast cancer. Genome Res. 22, 1995–2007 (2012).

    Article  CAS  Google Scholar 

  26. Rosenkrantz, J.L. & Carbone, L. Investigating somatic aneuploidy in the brain: why we need a new model. Chromosoma https:/dx.doi.org/10.1007/s00412-016-0615-4 (2016).

  27. Callaway, E. 'Platinum' genome takes on disease. Nature 515, 323 (2014).

    Article  CAS  Google Scholar 

  28. Waddell, N. et al. Whole genomes redefine the mutational landscape of pancreatic cancer. Nature 518, 495–501 (2015).

    Article  CAS  Google Scholar 

  29. De Kouchkovsky, I. & Abdul-Hay, M. Acute myeloid leukemia: a comprehensive review and 2016 update. Blood Cancer J. 6, e441 (2016).

    Article  CAS  Google Scholar 

  30. Kumagai, T. et al. Epigenetic regulation and molecular characterization of C/EBPalpha in pancreatic cancer cells. Int. J. Cancer 124, 827–833 (2009).

    Article  CAS  Google Scholar 

  31. Perkins, N.D. Integrating cell-signalling pathways with NF-kappaB and IKK function. Nat. Rev. Mol. Cell Biol. 8, 49–62 (2007).

    Article  CAS  Google Scholar 

  32. Stahley, S.N. & Kowalczyk, A.P. Desmosomes in acquired disease. Cell Tissue Res. 360, 439–456 (2015).

    Article  Google Scholar 

  33. Forbes, S.A. et al. COSMIC: exploring the world's knowledge of somatic mutations in human cancer. Nucleic Acids Res. 43, D805–D811 (2015).

    Article  CAS  Google Scholar 

  34. Bailey, P. et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52 (2016).

    Article  CAS  Google Scholar 

  35. Sos, B.C. et al. Characterization of chromatin accessibility with a transposome hypersensitive sites sequencing (THS-seq) assay. Genome Biol. 17, 20 (2016).

    Article  Google Scholar 

  36. Ramani, V. et al. Massively multiplex single-cell Hi-C. Nat. Methods http://dx.doi.org/10.1038/nmeth.4155 (2017).

  37. Vitak, S. et al. Sequencing thousands of single-cell genomes with combinatorial indexing. Protocol Exchange https://dx.doi.org/10.1038/protex.2017.001 (2017).

Download references

Acknowledgements

The genome sequence described and used in this research was derived from a HeLa cell line. Henrietta Lacks, and the HeLa cell line that was established from her tumor cells without her knowledge or consent in 1951, have made significant contributions to scientific progress and advances in human health. We are grateful to Henrietta Lacks, now deceased, and to her surviving family members for their contributions to biomedical research. The data generated from this research were submitted to the database of Genotypes and Phenotypes (dbGaP), as a substudy under accession number phs000640. We thank the aging nonhuman primate resource at the Oregon National Primate Research Center for the banked rhesus samples, the Brenden-Colson Center for Pancreatic Care for the pancreatic ductal adenocarcinoma sample, and the Knight Tissue Bank for the rectal adenocarcinoma sample. We thank J. Shendure and Shendure laboratory members D. Cusanovich and R. Daza for helpful advice and comments, and M. Kircher for providing PCR-stage index sequences. We also thank B.J. O'Roak and R. Mulqueen for helpful discussions and manuscript suggestions. A.A. is supported by an Oregon Medical Research Foundation New Investigator Award. J.L.R. is supported by the Collins Medical Trust Foundation and Glenn/AFAR Scholarship for Research in the Biology of Aging. L. Carbone is supported by the Office of the Director/Office of Research Infrastructure Programs (OD/ORIP) of the NIH (grant no. OD011092).

Author information

Author notes

  1. Sarah A Vitak and Kristof A Torkenczy: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular & Medical Genetics, Oregon Health & Science University, Portland, Oregon, USA

    Sarah A Vitak, Kristof A Torkenczy, Jimi L Rosenkrantz, Andrew J Fields, Lucia Carbone & Andrew Adey

  2. Program in Molecular & Cellular Biosciences, Oregon Health & Science University, Portland, Oregon, USA

    Kristof A Torkenczy & Jimi L Rosenkrantz

  3. Oregon National Primate Research Center, Beaverton, Oregon, USA

    Jimi L Rosenkrantz & Lucia Carbone

  4. Advanced Research Group, Illumina Inc., San Diego, California, USA

    Lena Christiansen & Frank J Steemers

  5. Department of Cell, Developmental & Cancer Biology, Oregon Health & Science University, Portland, Oregon, USA

    Melissa H Wong

  6. Knight Cancer Institute, Portland, Oregon, USA

    Melissa H Wong

  7. Department of Behavioral Neurosciences, Oregon Health & Science University, Portland, Oregon, USA

    Lucia Carbone

  8. Knight Cardiovascular Institute, Portland, Oregon, USA

    Lucia Carbone & Andrew Adey

Authors
  1. Sarah A Vitak
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kristof A Torkenczy
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jimi L Rosenkrantz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrew J Fields
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Lena Christiansen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Melissa H Wong
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Lucia Carbone
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Frank J Steemers
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Andrew Adey
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

A.A. designed and supervised all aspects of the study. A.A., S.A.V. and K.A.T. wrote the manuscript. All authors contributed to and edited the manuscript. S.A.V. carried out all SCI-seq and GM12878 DOP library preparations, designed experiments, and performed all sequencing. A.A. and K.A.T. processed all sequence data and analyzed data. K.A.T. performed all copy-number calling. J.L.R. constructed QRP and DOP libraries on rhesus samples. A.J.F. prepared all GM12878 QRP library construction and co-prepared all SCI-seq libraries using xSDS for nucleosome depletion. M.H.W. provided tumor samples and aided in the analyses of those samples. L. Carbone supervised and provided all samples for rhesus work. F.J.S. contributed to experimental design and contributed to the manuscript. L. Christiansen produced all transposase complexes used in this study.

Corresponding author

Correspondence to Andrew Adey.

Ethics declarations

Competing interests

F.J.S. and L. Christiansen declare competing financial interests in the form of paid employment by Illumina, Inc. One or more embodiments of one or more patents and patent applications filed by Illumina may encompass the methods, reagents, and data disclosed in this manuscript. Some work in this study is related to technology described in patent applications WO2014142850, 2014/0194324, 2010/0120098, 2011/0287435, 2013/0196860 and 2012/0208705. A.A. and S.A.V. have a provisional patent filed for some of the methods pertaining to this study.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–3, Supplementary Figures 1–25 and Supplementary Protocol (PDF 6652 kb)

Supplementary Software

Software for processing SCI-seq sequence read data. (ZIP 9 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Vitak, S., Torkenczy, K., Rosenkrantz, J. et al. Sequencing thousands of single-cell genomes with combinatorial indexing. Nat Methods 14, 302–308 (2017). https://doi.org/10.1038/nmeth.4154

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth.4154

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer