Cargando…

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

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....

Descripción completa

Detalles Bibliográficos
Autores principales: Rallapalli, Ghanasyam, Kemen, Eric M, Robert-Seilaniantz, Alexandre, Segonzac, Cécile, Etherington, Graham J, Sohn, Kee Hoon, MacLean, Daniel, Jones, Jonathan D G
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2014
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035070/
https://www.ncbi.nlm.nih.gov/pubmed/24884414
http://dx.doi.org/10.1186/1471-2164-15-341
_version_ 1782318017022001152
author Rallapalli, Ghanasyam
Kemen, Eric M
Robert-Seilaniantz, Alexandre
Segonzac, Cécile
Etherington, Graham J
Sohn, Kee Hoon
MacLean, Daniel
Jones, Jonathan D G
author_facet Rallapalli, Ghanasyam
Kemen, Eric M
Robert-Seilaniantz, Alexandre
Segonzac, Cécile
Etherington, Graham J
Sohn, Kee Hoon
MacLean, Daniel
Jones, Jonathan D G
author_sort Rallapalli, Ghanasyam
collection PubMed
description 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-341) contains supplementary material, which is available to authorized users.
format Online
Article
Text
id pubmed-4035070
institution National Center for Biotechnology Information
language English
publishDate 2014
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-40350702014-06-06 EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics Rallapalli, Ghanasyam Kemen, Eric M Robert-Seilaniantz, Alexandre Segonzac, Cécile Etherington, Graham J Sohn, Kee Hoon MacLean, Daniel Jones, Jonathan D G BMC Genomics Methodology Article 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/1471-2164-15-341) contains supplementary material, which is available to authorized users. BioMed Central 2014-05-06 /pmc/articles/PMC4035070/ /pubmed/24884414 http://dx.doi.org/10.1186/1471-2164-15-341 Text en © Rallapalli et al.; licensee BioMed Central Ltd. 2014 This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Methodology Article
Rallapalli, Ghanasyam
Kemen, Eric M
Robert-Seilaniantz, Alexandre
Segonzac, Cécile
Etherington, Graham J
Sohn, Kee Hoon
MacLean, Daniel
Jones, Jonathan D G
EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics
title EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics
title_full EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics
title_fullStr EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics
title_full_unstemmed EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics
title_short EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics
title_sort exprss: an illumina based high-throughput expression-profiling method to reveal transcriptional dynamics
topic Methodology Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4035070/
https://www.ncbi.nlm.nih.gov/pubmed/24884414
http://dx.doi.org/10.1186/1471-2164-15-341
work_keys_str_mv AT rallapallighanasyam exprssanilluminabasedhighthroughputexpressionprofilingmethodtorevealtranscriptionaldynamics
AT kemenericm exprssanilluminabasedhighthroughputexpressionprofilingmethodtorevealtranscriptionaldynamics
AT robertseilaniantzalexandre exprssanilluminabasedhighthroughputexpressionprofilingmethodtorevealtranscriptionaldynamics
AT segonzaccecile exprssanilluminabasedhighthroughputexpressionprofilingmethodtorevealtranscriptionaldynamics
AT etheringtongrahamj exprssanilluminabasedhighthroughputexpressionprofilingmethodtorevealtranscriptionaldynamics
AT sohnkeehoon exprssanilluminabasedhighthroughputexpressionprofilingmethodtorevealtranscriptionaldynamics
AT macleandaniel exprssanilluminabasedhighthroughputexpressionprofilingmethodtorevealtranscriptionaldynamics
AT jonesjonathandg exprssanilluminabasedhighthroughputexpressionprofilingmethodtorevealtranscriptionaldynamics