A study of the relationships between oligonucleotide properties and hybridization signal intensities from NimbleGen microarray datasets.
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 (T(m)) 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.
Wei
Hairong
H
WiCell Research Institute, PO Box 7365, Madison, WI 53707-7365, USA.
Kuan
Pei Fen
PF
Tian
Shulan
S
Yang
Chuhu
C
Nie
Jeff
J
Sengupta
Srikumar
S
Ruotti
Victor
V
Jonsdottir
Gudrun A
GA
Keles
Sunduz
S
Thomson
James A
JA
Stewart
Ron
R
D020869:Gene Expression Profiling; D006801:Humans; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes / Q000737:chemistry*; D012044:Regression Analysis; D012689:Sequence Homology, Nucleic Acid; D013696:Temperature
D015345:Oligonucleotide Probes