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Nat Protoc. 2016 Feb;11(2):214-235. doi: 10.1038/nprot.2016.005. Epub 2016 Jan 7.

Highly multiplexed targeted DNA sequencing from single nuclei.

Leung ML#1,2, Wang Y#1, Kim C1,2, Gao R1, Jiang J1, Sei E1, Navin NE1,2,3.

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Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Graduate Program in Genes and Development, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, Texas, USA.
Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
Contributed equally


Single-cell DNA sequencing methods are challenged by poor physical coverage, high technical error rates and low throughput. To address these issues, we developed a single-cell DNA sequencing protocol that combines flow-sorting of single nuclei, time-limited multiple-displacement amplification (MDA), low-input library preparation, DNA barcoding, targeted capture and next-generation sequencing (NGS). This approach represents a major improvement over our previous single nucleus sequencing (SNS) Nature Protocols paper in terms of generating higher-coverage data (>90%), thereby enabling the detection of genome-wide variants in single mammalian cells at base-pair resolution. Furthermore, by pooling 48-96 single-cell libraries together for targeted capture, this approach can be used to sequence many single-cell libraries in parallel in a single reaction. This protocol greatly reduces the cost of single-cell DNA sequencing, and it can be completed in 5-6 d by advanced users. This single-cell DNA sequencing protocol has broad applications for studying rare cells and complex populations in diverse fields of biological research and medicine.

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