Abstract

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumor's major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.

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Acknowledgements

The authors thank D. Adams, T. Whipp, R. Rance and the Wellcome Trust Sanger Institute sequencing and informatics teams for 454 sequencing; the Institute for Molecular and Cell Biology Histopathology Core; P. Cheok, N. Lim, D. Chen and C. Wee for assistance with tumor monitoring and animal husbandry at IMCB (Singapore), H. Lee and E. Freiter for assistance with animal husbandry at HMRI (Houston), R. Zahr (Integrated DNA Technologies, Inc.) for assistance with SBCapture probe design and D. Adams and C. Print for valuable discussions and critical reading of the manuscript. Histology work was performed by the Advanced Molecular Pathology Laboratory, IMCB, A*STAR. This work was supported by the Cancer Prevention Research Institute of Texas (N.G.C. and N.A.J.), the Biomedical Research Council, Agency for Science, Technology, and Research, Singapore (N.G.C. and N.A.J.), Cancer Research UK (A.G.R.), the Medical Research Council, UK (A.G.R.) and the Wellcome Trust (A.G.R.).

Author information

Author notes

    • Jerrold M Ward
    • , Alistair G Rust
    • , Christopher Chin Kuan Yew
    • , Jill L Waters
    •  & Luxmanan Selvanesan

    Present addresses: Global VetPathology, Montgomery Village, Maryland, USA (J.M.W.), Tumour Profiling Unit, the Institute of Cancer Research, Chester Beatty Laboratories, London, UK (A.G.R.), National Heart Research Institute Singapore, Republic of Singapore (C.C.K.Y.), Illumina, Inc., San Diego, California, USA (J.L.W.) and Pacific Edge Limited, Dunedin, Otago, New Zealand (L.S.).

    • Karen M Mann
    •  & Michael B Mann

    These authors contributed equally to this work.

Affiliations

  1. Cancer Research Program, Houston Methodist Research Institute, Houston, Texas, USA.

    • Karen M Mann
    • , Justin Y Newberg
    • , Devin J Jones
    • , Felipe Amaya-Manzanares
    • , Liliana Guzman-Rojas
    • , Takahiro Kodama
    • , Nancy A Jenkins
    • , Neal G Copeland
    •  & Michael B Mann
  2. Institute of Molecular and Cell Biology, Singapore, Republic of Singapore.

    • Karen M Mann
    • , Jerrold M Ward
    • , Christopher Chin Kuan Yew
    • , Keith Rogers
    • , Susan M Rogers
    • , Nancy A Jenkins
    • , Neal G Copeland
    •  & Michael B Mann
  3. Department of Biochemistry, University of Otago, Dunedin, New Zealand.

    • Michael A Black
    • , Leslie A McNoe
    •  & Luxmanan Selvanesan
  4. Experimental Cancer Genetics, Wellcome Trust Sanger Institute, Hinxton, Cambridge, UK.

    • Alistair G Rust
    •  & Louise van der Weyden
  5. Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas, USA.

    • Jill L Waters
    • , Marco L Leung
    •  & Nicholas Navin

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Contributions

K.M.M., M.B.M., N.G.C. and N.A.J. designed the study, directed the research, interpreted the data and wrote the manuscript. K.M.M. and M.B.M. performed experimental work, designed the SBCapSeq oligos, coordinated sequencing efforts and analyzed the data. M.A.B., J.M.W., A.G.R. and N.N. contributed to the experimental design. J.Y.N., M.A.B. and A.G.R. provided essential statistical and bioinformatics resources. J.Y.N. wrote the Python code for SBCapSeq and splink HiSeq workflows and gCIS analysis; performed statistical and bioinformatic analysis for SBCapSeq, splink HiSeq, and RNA-seq data; and managed compute resources and data archiving of SBCapSeq and splink HiSeq data. M.A.B. wrote the R code and performed statistical and bioinformatic analysis for microarray, RNA-seq and WGS data analysis. A.G.R. wrote the R and Perl code for splink 454 workflow and managed resources and data archiving of splink 454 data. K.R. and S.M.R. performed and directed necropsy and histopathological analysis. J.M.W. performed and directed veterinary pathology analysis, including tumor grading and diagnosis. L.v.d.W. optimized library preparation for splink 454 sequencing. C.C.K.Y. performed bioinformatic analysis for splink 454 data. L.A.M. and L.S. isolated RNA and performed microarray hybridizations. K.M.M. developed and optimized staining protocols for FACS analysis. J.L.W., M.L.L. and N.N. isolated single cells by FACS and performed WGA of single-cell genomes. D.J.J. performed and optimized library preparation for SBCapSeq method and performed library preparation for splink HiSeq sequencing. L.G.-R. and F.A.-M. performed and optimized capture hybridizations for the SBCapSeq method and performed Ion Torrent sequencing for SBCapSeq, RNA-seq and WGS experiments. T.K. optimized library preparation splink HiSeq sequencing. All coauthors contributed to editing the manuscript before submission. N.G.C. and N.A.J. provided laboratory resources and personnel for animal husbandry, specimen archiving, sequencing and computer management.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Neal G Copeland or Michael B Mann.

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https://doi.org/10.1038/nbt.3637

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