Journal Article

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

Sohn, Kee Hoon, Jones, Jonathan D G, Etherington, Graham J, Segonzac, Cécile, Rallapalli, Ghanasyam, MacLean, Daniel, Kemen, Eric M, Robert-Seilaniantz, Alexandre

2014

Overview

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, Nla III-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.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V
• Subject: 3' Untranslated Regions / Analysis / Animal Genetics and Genomics / Arabidopsis - genetics / Binomial distribution / Biomedical and Life Sciences / Charitable foundations / Computational Biology - methods / Data analysis / Deoxyribonucleic acid / DNA / DNA methylation / DNA, Complementary - metabolism / Down-Regulation / Enzymes / Gene expression / Gene Expression Profiling - methods / Gene Library / Genetic aspects / Genomes / Genomics / High-Throughput Nucleotide Sequencing / Investigations / Life Sciences / Messenger RNA / Methodology / Methodology Article / Methods / Microarrays / Microbial Genetics and Genomics / Oligonucleotide Array Sequence Analysis / Plant Genetics and Genomics / Proteomics / Protocol / Research parks / RNA, Messenger - genetics / Sequence Analysis, RNA - methods / Transcriptomic methods / Up-Regulation