Open Access Highly Accessed Methodology article

EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics

Ghanasyam Rallapalli1, Eric M Kemen12, Alexandre Robert-Seilaniantz13, Cécile Segonzac14, Graham J Etherington1, Kee Hoon Sohn14, Daniel MacLean1 and Jonathan D G Jones1*

Author Affiliations

1 The Sainsbury Laboratory, Norwich Research Park, Colney, Norwich, UK NR4 7UH

2 Max-Planck Institute for Plant Breeding Research, Carl-von-Linné-Weg 10, 50829 Köln, Germany

3 UMR INRA-Agrocampus Ouest-Université de Rennes 1, INRA, UMR1349, IGEPP, BP35327, 35653 Le Rheu Cedex, France

4 Institute of Agriculture and Environment, Massey University, Palmerston North 4442, New Zealand

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BMC Genomics 2014, 15:341  doi:10.1186/1471-2164-15-341

Published: 6 May 2014

Abstract

Background

Next Generation Sequencing technologies have facilitated differential gene expression analysis through RNA-seq and Tag-seq methods. RNA-seq has biases associated with transcript lengths, lacks uniform coverage of regions in mRNA and requires 10–20 times more reads than a typical Tag-seq. Most existing Tag-seq methods either have biases or not high throughput due to use of restriction enzymes or enzymatic manipulation of 5’ ends of mRNA or use of RNA ligations.

Results

We have developed EXpression Profiling through Randomly Sheared cDNA tag Sequencing (EXPRSS) that employs acoustic waves to randomly shear cDNA and generate sequence tags at a relatively defined position (~150-200 bp) from the 3′ end of each mRNA. Implementation of the method was verified through comparative analysis of expression data generated from EXPRSS, NlaIII-DGE and Affymetrix microarray and through qPCR quantification of selected genes. EXPRSS is a strand specific and restriction enzyme independent tag sequencing method that does not require cDNA length-based data transformations. EXPRSS is highly reproducible, is high-throughput and it also reveals alternative polyadenylation and polyadenylated antisense transcripts. It is cost-effective using barcoded multiplexing, avoids the biases of existing SAGE and derivative methods and can reveal polyadenylation position from paired-end sequencing.

Conclusions

EXPRSS Tag-seq provides sensitive and reliable gene expression data and enables high-throughput expression profiling with relatively simple downstream analysis.

Keywords:
Next generation sequencing; Tag-seq; High throughput expression profiling; RNA-seq; EXPRSS