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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...

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Autores principales: Ekblom, Robert, Slate, Jon, Horsburgh, Gavin J., Birkhead, Tim, Burke, Terry
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2012
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.
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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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