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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.

Author information

1
Department of Genetics, University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
2
Graduate Program in Genes and Development, Graduate School of Biomedical Sciences, University of Texas Health Science Center at Houston, Houston, Texas, USA.
3
Department of Bioinformatics and Computational Biology, University of Texas MD Anderson Cancer Center, Houston, Texas, USA.
#
Contributed equally

Abstract

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.

PMID:
26741407
PMCID:
PMC4807405
DOI:
10.1038/nprot.2016.005
[Indexed for MEDLINE]
Free PMC Article

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