Quantification of the yeast transcriptome by single-molecule sequencing.
Nat Biotechnol
; 27(7): 652-8, 2009 Jul.
Article
em En
| MEDLINE
| ID: mdl-19581875
ABSTRACT
We present single-molecule sequencing digital gene expression (smsDGE), a high-throughput, amplification-free method for accurate quantification of the full range of cellular polyadenylated RNA transcripts using a Helicos Genetic Analysis system. smsDGE involves a reverse-transcription and polyA-tailing sample preparation procedure followed by sequencing that generates a single read per transcript. We applied smsDGE to the transcriptome of Saccharomyces cerevisiae strain DBY746, using 6 of the available 50 channels in a single sequencing run, yielding on average 12 million aligned reads per channel. Using spiked-in RNA, accurate quantitative measurements were obtained over four orders of magnitude. High correlation was demonstrated across independent flow-cell channels, instrument runs and sample preparations. Transcript counting in smsDGE is highly efficient due to the representation of each transcript molecule by a single read. This efficiency, coupled with the high throughput enabled by the single-molecule sequencing platform, provides an alternative method for expression profiling.
Texto completo:
1
Bases de dados:
MEDLINE
Assunto principal:
Saccharomyces cerevisiae
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Análise de Sequência de DNA
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Perfilação da Expressão Gênica
Tipo de estudo:
Prognostic_studies
Idioma:
En
Revista:
Nat Biotechnol
Assunto da revista:
BIOTECNOLOGIA
Ano de publicação:
2009
Tipo de documento:
Article
País de afiliação:
Estados Unidos