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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.

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Figure 1: Sample preparation, sequencing and analysis workflow.
Figure 2: Data description.
Figure 3: Reproducibility and counting accuracy.
Figure 4: Count reproducibility.
Figure 5: TSS mapping.
Figure 6: Sequence information.

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Acknowledgements

We thank all of the past and present colleagues at Helicos who have contributed to this work.

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Correspondence to Tal Raz.

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All of the authors are or have been employees of Helicos Biosciences.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–3, Supplementary Tables 4 and 6, and Supplementary Methods (PDF 369 kb)

Supplementary Table 1

Transcript counts (XLS 1784 kb)

Supplementary Table 2

qPCR measurements (XLS 25 kb)

Supplementary Table 3

Detected sequence variants (XLS 498 kb)

Supplementary Table 5

Coverage peaks in yeast genome. (XLS 106 kb)

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Lipson, D., Raz, T., Kieu, A. et al. Quantification of the yeast transcriptome by single-molecule sequencing. Nat Biotechnol 27, 652–658 (2009). https://doi.org/10.1038/nbt.1551

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