Low-bias, Strand-specific Transcriptome Illumina Sequencing by On-flowcell Reverse Transcription (FRT-seq)

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2011 Oct 22

PMID 22015844

Citations 13

Authors

Lira Mamanova

Daniel J Turner

Affiliations

Soon will be listed here.

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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