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Comparative and Functional Genomics
Volume 2012, Article ID 281693, 8 pages
Research Article

Comparison between Normalised and Unnormalised 454-Sequencing Libraries for Small-Scale RNA-Seq Studies

1Department of Ecology and Genetics, Uppsala University, Norbyvägen 18 D, 75236 Uppsala, Sweden
2Department of Animal and Plant Sciences, University of Sheffield, Sheffield S10 2TN, UK

Received 15 September 2011; Accepted 25 October 2011

Academic Editor: Giulia Piaggio

Copyright © 2012 Robert Ekblom et al. 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.


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.