Nucleic Acids Research Advance Access published online on April 1, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn133
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Computational Biology |
A study of the relationships between oligonucleotide properties and hybridization signal intensities from NimbleGen microarray datasets
1WiCell Research Institute, PO Box 7365, Madison, WI 53707-7365, 2Department of Statistics, Medical Sciences Center, University of Wisconsin-Madison 1300 University Avenue, Madison, WI 53706, 3Genome Center of Wisconsin, University of Wisconsin-Madison, 425 Henry Mall, Madison, WI 53706-1580, 4Wisconsin National Primate Research Center, University of Wisconsin-Madison, 1220 Capitol Court, Madison, WI 53715-1299, 5Department of Anatomy, University of Wisconsin School of Medicine and Public Health, 470 N. Charter Street, Madison, WI 53706-1509 and 6Department of Biostatistics and Medical Informatics, University of Wisconsin-Madison, 600 Highland Avenue, Madison, WI 53706, USA
*To whom correspondence should be addressed. Tel: +1 608 890 0676; Fax: +1 608 890 0181; Email: ron{at}wicell.org
Received October 17, 2007. Revised February 15, 2008. Accepted March 10, 2008.
Well-defined relationships between oligonucleotide properties and hybridization signal intensities (HSI) can aid chip design, data normalization and true biological knowledge discovery. We clarify these relationships using the data from two microarray experiments containing over three million probes from 48 high-density chips. We find that melting temperature (Tm) has the most significant effect on HSI while length for the long oligonucleotides studied has very little effect. Analysis of positional effect using a linear model provides evidence that the protruding ends of probes contribute more than tethered ends to HSI, which is further validated by specifically designed match fragment sliding and extension experiments. The impact of sequence similarity (SeqS) on HSI is not significant in comparison with other oligonucleotide properties. Using regression and regression tree analysis, we prioritize these oligonucleotide properties based on their effects on HSI. The implications of our discoveries for the design of unbiased oligonucleotides are discussed. We propose that isothermal probes designed by varying the length is a viable strategy to reduce sequence bias, though imposing selection constraints on other oligonucleotide properties is also essential.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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