Quantification of the yeast transcriptome by single-molecule sequencing

Doron Lipson, Tal Raz*, Alix Kieu, Daniel R. Jones, Eldar Giladi, Edward Thayer, John F. Thompson, Stan Letovsky, Patrice Milos, Marie Causey

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

146 Scopus citations

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.

Original languageAmerican English
Pages (from-to)652-658
Number of pages7
JournalNature Biotechnology
Volume27
Issue number7
DOIs
StatePublished - Jul 2009
Externally publishedYes

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