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....
Autores principales: | , , , , , , , |
---|---|
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 |