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Sequencing thousands of single-cell genomes with combinatorial indexing

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.

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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).

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Sequence Read Archive

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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).

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Authors and Affiliations

Authors

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.

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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)

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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

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