Mismatch and G-stack modulated probe signals on SNP microarrays.
BACKGROUND
Single nucleotide polymorphism (SNP) arrays are important tools widely used for genotyping and copy number estimation. This technology utilizes the specific affinity of fragmented DNA for binding to surface-attached oligonucleotide DNA probes. We analyze the variability of the probe signals of Affymetrix GeneChip SNP arrays as a function of the probe sequence to identify relevant sequence motifs which potentially cause systematic biases of genotyping and copy number estimates.


RESULTS
The probe design of GeneChip SNP arrays enables us to disentangle different sources of intensity modulations such as the number of mismatches per duplex, matched and mismatched base pairings including nearest and next-nearest neighbors and their position along the probe sequence. The effect of probe sequence was estimated in terms of triple-motifs with central matches and mismatches which include all 256 combinations of possible base pairings. The probe/target interactions on the chip can be decomposed into nearest neighbor contributions which correlate well with free energy terms of DNA/DNA-interactions in solution. The effect of mismatches is about twice as large as that of canonical pairings. Runs of guanines (G) and the particular type of mismatched pairings formed in cross-allelic probe/target duplexes constitute sources of systematic biases of the probe signals with consequences for genotyping and copy number estimates. The poly-G effect seems to be related to the crowded arrangement of probes which facilitates complex formation of neighboring probes with at minimum three adjacent G's in their sequence.


CONCLUSIONS
The applied method of "triple-averaging" represents a model-free approach to estimate the mean intensity contributions of different sequence motifs which can be applied in calibration algorithms to correct signal values for sequence effects. Rules for appropriate sequence corrections are suggested.
Binder
Hans
H
Interdisciplinary Centre for Bioinformatics, Universität Leipzig, Leipzig, Germany. binder@izbi.uni-leipzig.de
Fasold
Mario
M
Glomb
Torsten
T
D000465:Algorithms; D000483:Alleles; D020816:Amino Acid Motifs; D020137:Base Pair Mismatch; D002138:Calibration; D004247:DNA / Q000235:genetics; D015342:DNA Probes / Q000235:genetics; D005838:Genotype; D006147:Guanine / Q000737:chemistry; D006720:Homozygote; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D015345:Oligonucleotide Probes / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide; D013816:Thermodynamics
D015342:DNA Probes; D015345:Oligonucleotide Probes; D006147:Guanine; D004247:DNA