PCR is an inherently biased process, which decreases the efficiency of transcriptome sequencing 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.
- Mamanova L, Turner DJ. (2011) Low-bias, strand-specific transcriptome Illumina sequencing by on-flowcell reverse transcription (FRT-seq). Nat Protoc 6(11), 1736-47. [abstract]