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Nucleic Acids Research Advance Access first published online on October 15, 2008
This version published online on October 31, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn705
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© 2008 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Deep sequencing-based expression analysis shows major advances in robustness, resolution and inter-lab portability over five microarray platforms

Peter A. C. 't Hoen1,*, Yavuz Ariyurek1, Helene H. Thygesen1, Erno Vreugdenhil2, Rolf H. A. M. Vossen1, Renée X. de Menezes1, Judith M. Boer1, Gert-Jan B. van Ommen1 and Johan T. den Dunnen1

1The Center for Human and Clinical Genetics and the Leiden Genome Technology Center, Leiden University Medical Center and 2The Department of Medical Pharmacology from the Leiden/Amsterdam Center for Drug Research, Leiden, The Netherlands

*To whom correspondence should be addressed. Tel: +31 71 526 9421; Fax: +31 71 526 8285; Email: p.a.c.hoen{at}lumc.nl

Received August 12, 2008. Revised September 16, 2008. Accepted September 29, 2008.

The hippocampal expression profiles of wild-type mice and mice transgenic for {delta}C-doublecortin-like kinase were compared with Solexa/Illumina deep sequencing technology and five different microarray platforms. With Illumina's digital gene expression assay, we obtained ~2.4 million sequence tags per sample, their abundance spanning four orders of magnitude. Results were highly reproducible, even across laboratories. With a dedicated Bayesian model, we found differential expression of 3179 transcripts with an estimated false-discovery rate of 8.5%. This is a much higher figure than found for microarrays. The overlap in differentially expressed transcripts found with deep sequencing and microarrays was most significant for Affymetrix. The changes in expression observed by deep sequencing were larger than observed by microarrays or quantitative PCR. Relevant processes such as calmodulin-dependent protein kinase activity and vesicle transport along microtubules were found affected by deep sequencing but not by microarrays. While undetectable by microarrays, antisense transcription was found for 51% of all genes and alternative polyadenylation for 47%. We conclude that deep sequencing provides a major advance in robustness, comparability and richness of expression profiling data and is expected to boost collaborative, comparative and integrative genomics studies.


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