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Scalable whole-genome single-cell library preparation without preamplification

Abstract

Single-cell genomics is critical for understanding cellular heterogeneity in cancer, but existing library preparation methods are expensive, require sample preamplification and introduce coverage bias. Here we describe direct library preparation (DLP), a robust, scalable, and high-fidelity method that uses nanoliter-volume transposition reactions for single-cell whole-genome library preparation without preamplification. We examined 782 cells from cell lines and triple-negative breast xenograft tumors. Low-depth sequencing, compared with existing methods, revealed greater coverage uniformity and more reliable detection of copy-number alterations. Using phylogenetic analysis, we found minor xenograft subpopulations that were undetectable by bulk sequencing, as well as dynamic clonal expansion and diversification between passages. Merging single-cell genomes in silico, we generated 'bulk-equivalent' genomes with high depth and uniform coverage. Thus, low-depth sequencing of DLP libraries may provide an attractive replacement for conventional bulk sequencing methods, permitting analysis of copy number at the cell level and of other genomic variants at the population level.

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Figure 1: Single-cell genome analysis with DLP.
Figure 2: Coverage uniformity and sequencing metrics.
Figure 3: Single-cell copy-number profiles from xenograft SA501X3F.
Figure 4: Analysis of merged clonal genomes for xenograft SA501X3F.
Figure 5: Analysis of SNVs, LOH, and breakpoints for xenograft SA501X3F.

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Acknowledgements

We gratefully acknowledge funding support from the BC Cancer Foundation, the Canadian Breast Cancer Foundation, Genome Canada/Genome BC, the Natural Sciences & Engineering Research Council of Canada (grant RGPIN 386152-10 to C.L.H.), the Terry Fox Research Institute (grant NFP 1021 to S.A. and S.P.S.), the Canadian Institutes of Health Research (grant MOP 126119 to S.A. and S.P.S.), and the Canadian Cancer Society Research Institute (grant 701584 to S.A. and S.P.S.). S.A. and S.P.S. are supported as Canada Research Chairs, and S.P.S. is supported as a Michael Smith Foundation for Health Research Scholar. H.Z. and A.S. are each supported by a Vanier Canada Graduate Scholarship.

Author information

Authors and Affiliations

Authors

Contributions

H.Z., A.S., S.P.S., S.A., and C.L.H. designed the research. H.Z. performed experiments. A.S. analyzed the data. A.S., H.Z., C.L.H., S.A., and S.P.S. wrote the paper. E.L. prepared tissue samples and bulk libraries. P.E. performed xenograft transplants. M.V. contributed to technology development. C.L.H., S.A., and S.P.S. supervised the research.

Corresponding authors

Correspondence to Sohrab P Shah, Samuel Aparicio or Carl L Hansen.

Ethics declarations

Competing interests

C.L.H., H.Z., A.S., S.A. and S.P.S. are inventors on a patent application covering elements of the technology described here and have a financial interest through revenue-sharing policies of the University of British Columbia (UBC). C.L.H. has a financial interest in AbCellera, a company that has licensed rights from UBC to the aforementioned patent application.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–14, Supplementary Tables 1 and 6, and Supplementary Note (PDF 15838 kb)

Supplementary Table 2

DLP single-cell sequencing metrics for immortalized normal cell lines 184-hTERT-L2 (page 1) and GM18507 (page 2). (XLS 87 kb)

Supplementary Table 3

DLP single-cell sequencing metrics for patient-derived triple-negative breast cancer xenograft tumours SA501X3F (page 1) and SA501X4F (page 2). (XLS 170 kb)

Supplementary Table 4

Statistics table with Kruskal–Wallis tests (page 1) and Pearson's correlations (page 2). (XLS 22 kb)

Supplementary Table 5

Sequencing metrics for DLP merged bulk-equivalent and standard bulk genomes. (XLS 16 kb)

Supplementary Data

Microfluidic device AutoCAD design file. (ZIP 786 kb)

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Zahn, H., Steif, A., Laks, E. et al. Scalable whole-genome single-cell library preparation without preamplification. Nat Methods 14, 167–173 (2017). https://doi.org/10.1038/nmeth.4140

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