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Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies
Next-generation sequencing of transcriptomes (RNA-Seq) is being used increasingly in studies of nonmodel organisms. Here, we evaluate the effectiveness of normalising cDNA libraries prior to sequencing in a small-scale study of the zebra finch. We find that assemblies produced from normalised librar...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Hindawi Publishing Corporation
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272792/ https://www.ncbi.nlm.nih.gov/pubmed/22319409 http://dx.doi.org/10.1155/2012/281693 |
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author | Ekblom, Robert Slate, Jon Horsburgh, Gavin J. Birkhead, Tim Burke, Terry |
author_facet | Ekblom, Robert Slate, Jon Horsburgh, Gavin J. Birkhead, Tim Burke, Terry |
author_sort | Ekblom, Robert |
collection | PubMed |
description | Next-generation sequencing of transcriptomes (RNA-Seq) is being used increasingly in studies of nonmodel organisms. Here, we evaluate the effectiveness of normalising cDNA libraries prior to sequencing in a small-scale study of the zebra finch. We find that assemblies produced from normalised libraries had a larger number of contigs but used fewer reads compared to unnormalised libraries. Considerably more genes were also detected using the contigs produced from normalised cDNA, and microsatellite discovery was up to 73% more efficient in these. There was a positive correlation between the detected expression level of genes in normalised and unnormalised cDNA, and there was no difference in the number of genes identified as being differentially expressed between blood and spleen for the normalised and unnormalised libraries. We conclude that normalised cDNA libraries are preferable for many applications of RNA-Seq and that these can also be used in quantitative gene expression studies. |
format | Online Article Text |
id | pubmed-3272792 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | Hindawi Publishing Corporation |
record_format | MEDLINE/PubMed |
spelling | pubmed-32727922012-02-08 Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies Ekblom, Robert Slate, Jon Horsburgh, Gavin J. Birkhead, Tim Burke, Terry Comp Funct Genomics Research Article Next-generation sequencing of transcriptomes (RNA-Seq) is being used increasingly in studies of nonmodel organisms. Here, we evaluate the effectiveness of normalising cDNA libraries prior to sequencing in a small-scale study of the zebra finch. We find that assemblies produced from normalised libraries had a larger number of contigs but used fewer reads compared to unnormalised libraries. Considerably more genes were also detected using the contigs produced from normalised cDNA, and microsatellite discovery was up to 73% more efficient in these. There was a positive correlation between the detected expression level of genes in normalised and unnormalised cDNA, and there was no difference in the number of genes identified as being differentially expressed between blood and spleen for the normalised and unnormalised libraries. We conclude that normalised cDNA libraries are preferable for many applications of RNA-Seq and that these can also be used in quantitative gene expression studies. Hindawi Publishing Corporation 2012 2012-01-26 /pmc/articles/PMC3272792/ /pubmed/22319409 http://dx.doi.org/10.1155/2012/281693 Text en Copyright © 2012 Robert Ekblom et al. https://creativecommons.org/licenses/by/3.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Ekblom, Robert Slate, Jon Horsburgh, Gavin J. Birkhead, Tim Burke, Terry Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies |
title | Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies |
title_full | Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies |
title_fullStr | Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies |
title_full_unstemmed | Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies |
title_short | Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies |
title_sort | comparison between normalised and unnormalised 454-sequencing libraries for small-scale rna-seq studies |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3272792/ https://www.ncbi.nlm.nih.gov/pubmed/22319409 http://dx.doi.org/10.1155/2012/281693 |
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