Quantification of the yeast transcriptome by single-molecule sequencing.
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.
Lipson
Doron
D
Helicos Biosciences Corporation, Cambridge, Massachusetts, USA.
Raz
Tal
T
Kieu
Alix
A
Jones
Daniel R
DR
Giladi
Eldar
E
Thayer
Edward
E
Thompson
John F
JF
Letovsky
Stan
S
Milos
Patrice
P
Causey
Marie
M
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*
D012331:RNA, Fungal; D012333:RNA, Messenger