FRT-Seq

FRT-Seq

Flow-cell surface reverse transcription sequencing (FRT-Seq) is an transcriptome sequencing technique developed in 2010 by Mamanova et al. The method is strand-specific, free of amplification, and compatible with paired-end sequencing.

To begin with, poly(A) RNA samples are fragmented by metal-ion hydrolysis and dephosphorylated. Next, P7 primer/adapters are ligated to the 3' end of the fragments. The 3' adapter sequence starts at the 5' terminus with 20 nt of RNA followed by DNA nucleotides. The adapters are also 5' phosphorylated and blocked with dideoxycytosine (ddC) at the 3' end. After 3' adapter ligation, the fragments are size-selected for nucleotide fragments longer than the adapter. The 5' ends of the fragments are phosphorylated and ligated to P5 adapters. These adapters are blocked with an amino-C6 linker at the 5' end. Now that the fragments are flanked by adapters, they are hybridized to the flow cell and reverse-transcribed before cluster generation and sequencing.

Pros:

• Strand-specific poly(A) mRNA sequencing for transcriptome analysis
• No amplification step - gives more accurate representation of the total mRNA population, preventing amplification bias

Cons:

• Requires a large amount of input RNA material (250 ng)
• Selects only poly(A) mRNA samples

• FRT-Seq: Mamanova L., Andrews R. M., James K. D., Sheridan E. M., Ellis P. D., et al. (2010) FRT-seq: amplification-free, strand-specific transcriptome sequencing. Nat Methods 7: 130-132

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