title: Quantification of the yeast transcriptome by single-molecule sequencing.
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.
authors: Helicos Biosciences Corporation, Cambridge, Massachusetts, USA.
authors: Doron
authors: D
authors: Lipson
authors: Tal
authors: T
authors: Raz
authors: Alix
authors: A
authors: Kieu
authors: Daniel R
authors: DR
authors: Jones
authors: Eldar
authors: E
authors: Giladi
authors: Edward
authors: E
authors: Thayer
authors: John F
authors: JF
authors: Thompson
authors: Stan
authors: S
authors: Letovsky
authors: Patrice
authors: P
authors: Milos
authors: Marie
authors: M
authors: Causey
mesh_terms: D001483:Base Sequence; D002874:Chromosome Mapping; D020224:Expressed Sequence Tags; D020869:Gene Expression Profiling / Q000379:methods*; D016681:Genome, Fungal; D008969:Molecular Sequence Data; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D012331:RNA, Fungal; D012333:RNA, Messenger