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Clonal tracking using embedded viral barcoding and high-throughput sequencing

Nature Protocols volume 15, pages 1436–1458 (2020)Cite this article

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Abstract

Embedded viral barcoding in combination with high-throughput sequencing is a powerful technology with which to track single-cell clones. It can provide clonal-level insights into cellular proliferation, development, differentiation, migration, and treatment efficacy. Here, we present a detailed protocol for a viral barcoding procedure that includes the creation of barcode libraries, the viral delivery of barcodes, the recovery of barcodes, and the computational analysis of barcode sequencing data. The entire procedure can be completed within a few weeks. This barcoding method requires cells to be susceptible to viral transduction. It provides high sensitivity and throughput, and enables precise quantification of cellular progeny. It is cost efficient and does not require any advanced skills. It can also be easily adapted to many types of applications, including both in vitro and in vivo experiments.

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Fig. 1: Experiment workflow.
Fig. 2: Comparing barcode extraction replicates.
Fig. 3: qPCR amplification of barcode.
Fig. 4: Optimizing the edit distance thresholds.
Fig. 5: Python pipeline outputs.

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Data availability

A sample dataset has been deposited in Figshare: https://doi.org/10.35092/yhjc.11374446. This dataset was used to generate Figs. 4 and 5.

Code availability

The Python scripts have been provided in the Supplementary Software of this paper. The code in this paper has been peer-reviewed.

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Acknowledgements

We thank all members of the Lu lab for helping to optimize the protocol and C. Lytal for help editing the text. We thank the USC Stem Cell Flow Cytometry Facility and CHLA Sequencing Core for technical support. This research was funded primarily by a National Institutes of Health (NIH) R00 early investigator grant (NIH-R00-HL113104) and R01 grants (R01HL135292 and R01HL138225). R.L. is a Scholar of the Leukemia & Lymphoma Society and a Richard N. Merkin Assistant Professor. The project described was supported in part by award no. P30CA014089 from the National Cancer Institute. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Cancer Institute or the National Institutes of Health.

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Author notes

  1. These authors contributed equally: Charles Bramlett, Du Jiang.

Authors and Affiliations

  1. Eli and Edythe Broad CIRM Center for Regenerative Medicine and Stem Cell Research, Keck School of Medicine, University of Southern California, Los Angeles, Los Angeles, CA, USA

    Charles Bramlett, Du Jiang, Anna Nogalska, Jiya Eerdeng, Jorge Contreras & Rong Lu

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Contributions

R.L. conceived and developed the protocol. C.B., D.J., J.C., and A.N. optimized the barcode extraction protocol. D.J. and J.E. improved the Python code for analyzing high-throughput sequencing data. C.B., D.J., and R.L. prepared the manuscript. J.C. and A.N. provided assistance in manuscript text preparation.

Corresponding author

Correspondence to Rong Lu.

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The authors declare no competing interests.

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Peer review information Nature Protocols thanks Leïla Périé and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Related links

Key references using this protocol

Lu, R., Neff, N. R., Quake, S. R. & Weissman, I. L. Nat. Biotechnol. 29, 928–933 (2011): https://doi.org/10.1038/nbt.1977

Nguyen, L. et al. EMBO Rep. 19, e45702 (2018): https://doi.org/10.15252/embr.201745702

Brewer, C., Chu, E., Chin, M. & Lu, R. Cell Rep. 15, 1848–1857 (2016): https://doi.org/10.1016/j.celrep.2016.04.061

Supplementary information

Reporting Summary

Supplementary Software

The software for data analysis (Steps 121–127)

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Bramlett, C., Jiang, D., Nogalska, A. et al. Clonal tracking using embedded viral barcoding and high-throughput sequencing. Nat Protoc 15, 1436–1458 (2020). https://doi.org/10.1038/s41596-019-0290-z

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