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3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer
BACKGROUND: Massive parallel sequencing has the potential to replace microarrays as the method for transcriptome profiling. Currently there are two protocols: full-length RNA sequencing (RNA-SEQ) and 3'-tag digital gene expression (DGE). In this preliminary effort, we evaluated the 3' DGE...
Autores principales: | , , , , , , , , , , |
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Formato: | Texto |
Lenguaje: | English |
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BioMed Central
2009
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781828/ https://www.ncbi.nlm.nih.gov/pubmed/19917133 http://dx.doi.org/10.1186/1471-2164-10-531 |
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author | Asmann, Yan W Klee, Eric W Thompson, E Aubrey Perez, Edith A Middha, Sumit Oberg, Ann L Therneau, Terry M Smith, David I Poland, Gregory A Wieben, Eric D Kocher, Jean-Pierre A |
author_facet | Asmann, Yan W Klee, Eric W Thompson, E Aubrey Perez, Edith A Middha, Sumit Oberg, Ann L Therneau, Terry M Smith, David I Poland, Gregory A Wieben, Eric D Kocher, Jean-Pierre A |
author_sort | Asmann, Yan W |
collection | PubMed |
description | BACKGROUND: Massive parallel sequencing has the potential to replace microarrays as the method for transcriptome profiling. Currently there are two protocols: full-length RNA sequencing (RNA-SEQ) and 3'-tag digital gene expression (DGE). In this preliminary effort, we evaluated the 3' DGE approach using two reference RNA samples from the MicroArray Quality Control Consortium (MAQC). RESULTS: Using Brain RNA sample from multiple runs, we demonstrated that the transcript profiles from 3' DGE were highly reproducible between technical and biological replicates from libraries constructed by the same lab and even by different labs, and between two generations of Illumina's Genome Analyzers. Approximately 65% of all sequence reads mapped to mitochondrial genes, ribosomal RNAs, and canonical transcripts. The expression profiles of brain RNA and universal human reference RNA were compared which demonstrated that DGE was also highly quantitative with excellent correlation of differential expression with quantitative real-time PCR. Furthermore, one lane of 3' DGE sequencing, using the current sequencing chemistry and image processing software, had wider dynamic range for transcriptome profiling and was able to detect lower expressed genes which are normally below the detection threshold of microarrays. CONCLUSION: 3' tag DGE profiling with massive parallel sequencing achieved high sensitivity and reproducibility for transcriptome profiling. Although it lacks the ability of detecting alternative splicing events compared to RNA-SEQ, it is much more affordable and clearly out-performed microarrays (Affymetrix) in detecting lower abundant transcripts. |
format | Text |
id | pubmed-2781828 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2009 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-27818282009-11-25 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer Asmann, Yan W Klee, Eric W Thompson, E Aubrey Perez, Edith A Middha, Sumit Oberg, Ann L Therneau, Terry M Smith, David I Poland, Gregory A Wieben, Eric D Kocher, Jean-Pierre A BMC Genomics Research article BACKGROUND: Massive parallel sequencing has the potential to replace microarrays as the method for transcriptome profiling. Currently there are two protocols: full-length RNA sequencing (RNA-SEQ) and 3'-tag digital gene expression (DGE). In this preliminary effort, we evaluated the 3' DGE approach using two reference RNA samples from the MicroArray Quality Control Consortium (MAQC). RESULTS: Using Brain RNA sample from multiple runs, we demonstrated that the transcript profiles from 3' DGE were highly reproducible between technical and biological replicates from libraries constructed by the same lab and even by different labs, and between two generations of Illumina's Genome Analyzers. Approximately 65% of all sequence reads mapped to mitochondrial genes, ribosomal RNAs, and canonical transcripts. The expression profiles of brain RNA and universal human reference RNA were compared which demonstrated that DGE was also highly quantitative with excellent correlation of differential expression with quantitative real-time PCR. Furthermore, one lane of 3' DGE sequencing, using the current sequencing chemistry and image processing software, had wider dynamic range for transcriptome profiling and was able to detect lower expressed genes which are normally below the detection threshold of microarrays. CONCLUSION: 3' tag DGE profiling with massive parallel sequencing achieved high sensitivity and reproducibility for transcriptome profiling. Although it lacks the ability of detecting alternative splicing events compared to RNA-SEQ, it is much more affordable and clearly out-performed microarrays (Affymetrix) in detecting lower abundant transcripts. BioMed Central 2009-11-16 /pmc/articles/PMC2781828/ /pubmed/19917133 http://dx.doi.org/10.1186/1471-2164-10-531 Text en Copyright ©2009 Asmann et al; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research article Asmann, Yan W Klee, Eric W Thompson, E Aubrey Perez, Edith A Middha, Sumit Oberg, Ann L Therneau, Terry M Smith, David I Poland, Gregory A Wieben, Eric D Kocher, Jean-Pierre A 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer |
title | 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer |
title_full | 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer |
title_fullStr | 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer |
title_full_unstemmed | 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer |
title_short | 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer |
title_sort | 3' tag digital gene expression profiling of human brain and universal reference rna using illumina genome analyzer |
topic | Research article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2781828/ https://www.ncbi.nlm.nih.gov/pubmed/19917133 http://dx.doi.org/10.1186/1471-2164-10-531 |
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