Article abstract
Nature Biotechnology 27, 652 - 658 (2009)
Published online: 5 July 2009 | doi:10.1038/nbt.1551
Quantification of the yeast transcriptome by single-molecule sequencing
Doron Lipson1,2, Tal Raz1,2, Alix Kieu1, Daniel R Jones1, Eldar Giladi1, Edward Thayer1, John F Thompson1, Stan Letovsky1, Patrice Milos1 & Marie Causey1
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.
- Helicos Biosciences Corporation, Cambridge, Massachusetts, USA.
- These authors contributed equally to this work.
Correspondence to: Tal Raz1,2 e-mail: traz@helicosbio.com
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