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. 2010 Feb;7(2):130-2.
doi: 10.1038/nmeth.1417. Epub 2010 Jan 17.

FRT-seq: amplification-free, strand-specific transcriptome sequencing

Lira Mamanova  1 , Robert M Andrews, Keith D James, Elizabeth M Sheridan, Peter D Ellis, Cordelia F Langford, Tobias W B Ost, John E Collins, Daniel J Turner
Affiliations

FRT-seq: amplification-free, strand-specific transcriptome sequencing

Lira Mamanova et al. Nat Methods. 2010 Feb.

Abstract

We report an alternative approach to transcriptome sequencing for the Illumina Genome Analyzer, in which the reverse transcription reaction takes place on the flowcell. No amplification is performed during the library preparation, so PCR biases and duplicates are avoided, and because the template is poly(A)(+) RNA rather than cDNA, the resulting sequences are necessarily strand-specific. The method is compatible with paired- or single-end sequencing.

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Figures

Figure 1
Figure 1. Correlation plots for FRT-seq libraries
We plotted sequence data obtained from two FRT libraries prepared from the same poly A+ RNA sample. All reads were mapped to annotated genes from the ENSEMBL database, normalized read counts and calculated Pearson correlations between the libraries. RKPM = reads per kilobase of sequence per million reads.
Figure 2
Figure 2. Strand specificity of FRT-seq
Sequences generated by FRT-seq were mapped against the human genome,. .wig files are displayed in IGB, though the colours were modified for clarity (dark red). For comparison, sequences made using the standard RT-seq library preparation protocols and flowcell amplification are also shown (blue). Below is a representation of the region of human chromosome 1p36, and beneath this genes are shown in Ensembl together with the strands from which the transcript is produced.
See this image and copyright information in PMC

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