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

Nat Protoc. 2011 Oct 20;6(11):1736-47. doi: 10.1038/nprot.2011.399.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chromosome Mapping / methods
  • Fluorescence Resonance Energy Transfer / methods*
  • Gene Expression Profiling / methods*
  • Nucleic Acid Amplification Techniques / methods*
  • RNA, Messenger / chemistry*
  • Reverse Transcription*

Substances

  • RNA, Messenger