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.
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We thank all of the past and present colleagues at Helicos who have contributed to this work.
All of the authors are or have been employees of Helicos Biosciences.
Supplementary Figures 1–3, Supplementary Tables 4 and 6, and Supplementary Methods (PDF 369 kb)
Transcript counts (XLS 1784 kb)
qPCR measurements (XLS 25 kb)
Detected sequence variants (XLS 498 kb)
Coverage peaks in yeast genome. (XLS 106 kb)
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