Highly multiplexed targeted DNA sequencing from single nuclei.
Nat Protoc
; 11(2): 214-235, 2016 Feb.
Article
em En
| MEDLINE
| ID: mdl-26741407
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.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Núcleo Celular
/
Análise de Sequência de DNA
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Análise de Célula Única
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Sequenciamento de Nucleotídeos em Larga Escala
Idioma:
En
Revista:
Nat Protoc
Ano de publicação:
2016
Tipo de documento:
Article