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Low-bias, strand-specific transcriptome Illumina sequencing by on-flowcell reverse transcription (FRT-seq)

Nature Protocols volume 6, pages 1736–1747 (2011)Cite this article

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Abstract

The unifying feature of second-generation sequencing technologies is that single template strands are amplified clonally onto a solid surface prior to the sequencing reaction. To convert template strands into a compatible state for attachment to this surface, a multistep library preparation is required, which typically culminates in amplification by the PCR. PCR is an inherently biased process, which decreases the efficiency of data acquisition. Flowcell reverse transcription sequencing is a method of transcriptome sequencing for Illumina sequencers in which the reverse transcription reaction is performed on the flowcell by using unamplified, adapter-ligated mRNA as a template. This approach removes PCR biases and duplicates, generates strand-specific paired-end data and is highly reproducible. The procedure can be performed quickly, taking 2 d to generate clusters from mRNA.

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Figure 1: FRT-seq workflow.
Figure 2: Agilent Bioanalyzer 2100 RNA chip results for a fragmented RNA sample.
Figure 3: Removal of unligated adapter.
Figure 4: Agilent Bioanalyzer 2100 RNA chip results for a ligated RNA sample.
Figure 5: Reproducibility and strand specificity of FRT-seq.

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Acknowledgements

We thank R. Andrews, K. James, L. Sheridan, P. Ellis, C. Langford, T. Ost and J. Collins, who co-authored the primary paper, and M. Gibbs, who helped with the cBot recipes. This work was supported by the Wellcome Trust, grant no. WT079643.

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  1. Daniel J Turner

    Present address: Present address: Oxford Nanopore Technologies, Oxford, UK.,

Authors and Affiliations

  1. The Wellcome Trust Sanger Institute, Cambridge, UK

    Lira Mamanova & Daniel J Turner

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  1. Lira Mamanova
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Contributions

D.J.T. designed the study; L.M. developed protocols and conducted the experiments; D.J.T. and L.M. wrote the manuscript.

Corresponding author

Correspondence to Lira Mamanova.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Method 1

cBot_FRTseq_amp_v7.0.txt — recipe for reverse transcription and cluster amplification for both single and paired end flowcells on cBot (XML 3 kb)

Supplementary Method 2

cBot_PE_Destain_Lin_Block_Hyb_v7.0.txt — recipe for paired end read flowcells, destain, linearization and blocking on cBot (XML 5 kb)

Supplementary Method 3

cBot_SR_Destain_Lin_Block_Hyb_v7.0.txt — recipe for single read flowcells, destain, linearization and blocking on cBot (XML 5 kb)

Supplementary Method 4

CS_FRTseq_amp_v5.0.txt — recipe for reverse transcription and cluster amplification on Cluster Station (XML 10 kb)

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Mamanova, L., Turner, D. Low-bias, strand-specific transcriptome Illumina sequencing by on-flowcell reverse transcription (FRT-seq). Nat Protoc 6, 1736–1747 (2011). https://doi.org/10.1038/nprot.2011.399

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