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"Hook"-calibration of GeneChip-microarrays: chip characteristics and expression measures.

Binder H, Krohn K, Preibisch S - Algorithms Mol Biol (2008)

Bottom Line: We show that the proper judgement of these effects requires the disentanglement of non-specific and specific hybridization which, otherwise, can lead to misinterpretations of expression changes.The consequences of modifying probe/target interactions by either changing the labelling protocol or by substituting RNA by DNA targets are demonstrated.The single-chip based hook-method provides accurate expression estimates and chip-summary characteristics using the natural metrics given by the hybridization reaction with the potency to develop new standards for microarray quality control and calibration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Centre for Bioinformatics, University of Leipzig, D-04107 Leipzig, Germany. binder@izbi.uni-leipzig.de

ABSTRACT

Background: Microarray experiments rely on several critical steps that may introduce biases and uncertainty in downstream analyses. These steps include mRNA sample extraction, amplification and labelling, hybridization, and scanning causing chip-specific systematic variations on the raw intensity level. Also the chosen array-type and the up-to-dateness of the genomic information probed on the chip affect the quality of the expression measures. In the accompanying publication we presented theory and algorithm of the so-called hook method which aims at correcting expression data for systematic biases using a series of new chip characteristics.

Results: In this publication we summarize the essential chip characteristics provided by this method, analyze special benchmark experiments to estimate transcript related expression measures and illustrate the potency of the method to detect and to quantify the quality of a particular hybridization. It is shown that our single-chip approach provides expression measures responding linearly on changes of the transcript concentration over three orders of magnitude. In addition, the method calculates a detection call judging the relation between the signal and the detection limit of the particular measurement. The performance of the method in the context of different chip generations and probe set assignments is illustrated. The hook method characterizes the RNA-quality in terms of the 3'/5'-amplification bias and the sample-specific calling rate. We show that the proper judgement of these effects requires the disentanglement of non-specific and specific hybridization which, otherwise, can lead to misinterpretations of expression changes. The consequences of modifying probe/target interactions by either changing the labelling protocol or by substituting RNA by DNA targets are demonstrated.

Conclusion: The single-chip based hook-method provides accurate expression estimates and chip-summary characteristics using the natural metrics given by the hybridization reaction with the potency to develop new standards for microarray quality control and calibration.

No MeSH data available.


Related in: MedlinePlus

Hybridization ranges of the raw (lower part) and the corrected (upper part) hook-curves calculated from hybridizations of the HG-U95 (left) and DG-1 (right) Gene Chips (see also Figure 1). The dotted lines indicate the hybridization ranges characterized by predominantly non-specific (N) and specific (S) binding, by a mixture of significant S- and N-contributions (mix), by the progressive saturation of the probe spots with bound transcripts (sat) and by almost completely saturated probes (as). Affinity correction considerably changes the shape of the hook-curve and the extent of the hybridization ranges. The corrected hook-curve and the fit are characterized by their geometrical dimensions; width (β), height (~α), start- (Σ(0), Δ(0)) and end- (Σ(∞)) positions; which in turn characterize the particular hybridization in terms of the mean non-specific background contribution, the PM/MM-gain etc. (see Table 2 for details). Compare also with Figure 1: The HG-U95 data were taken from different experiment series (Affymetrix spiked-in series here [3] and Genelogic dilution series [1] in Figure 1).
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Figure 2: Hybridization ranges of the raw (lower part) and the corrected (upper part) hook-curves calculated from hybridizations of the HG-U95 (left) and DG-1 (right) Gene Chips (see also Figure 1). The dotted lines indicate the hybridization ranges characterized by predominantly non-specific (N) and specific (S) binding, by a mixture of significant S- and N-contributions (mix), by the progressive saturation of the probe spots with bound transcripts (sat) and by almost completely saturated probes (as). Affinity correction considerably changes the shape of the hook-curve and the extent of the hybridization ranges. The corrected hook-curve and the fit are characterized by their geometrical dimensions; width (β), height (~α), start- (Σ(0), Δ(0)) and end- (Σ(∞)) positions; which in turn characterize the particular hybridization in terms of the mean non-specific background contribution, the PM/MM-gain etc. (see Table 2 for details). Compare also with Figure 1: The HG-U95 data were taken from different experiment series (Affymetrix spiked-in series here [3] and Genelogic dilution series [1] in Figure 1).

Mentions: Figure 1 depicts a typical graphical output-summary of the hook-analysis for two hybridizations performed on two different chip-types taken from the Genelogic dilution [1] and the GoldenSpike [2] experimental series (see also Figure 2 with data taken from the HG-U95 Latin square spiked-in series [3]). The Δ-vs-Σ plots characterize the hybridization of the particular chip. They are obtained by transforming the probe intensities of one GeneChip microarray into Δ = logIPM - logIMM and Σ = 0.5(logIPM + logIMM) coordinates and subsequent smoothing (IPM and IMM denote the spot intensities of the PM and MM probes after optical background correction; the logs are base 10 throughout the paper). The corrected version of the Δ-vs-Σ plot uses intensity values which are corrected for sequence-specific sensitivity effects. These plots are called hook-curves because of their typical shape. Additional characteristics of a particular chip-hybridization are the signal-density distribution and the four positional-dependent sensitivity profiles of the PM and MM probes upon specific and non-specific hybridization, respectively. These profiles are calculated from the intensity data of the chosen chip and used to correct the intensities for sequence-specific affinities.


"Hook"-calibration of GeneChip-microarrays: chip characteristics and expression measures.

Binder H, Krohn K, Preibisch S - Algorithms Mol Biol (2008)

Hybridization ranges of the raw (lower part) and the corrected (upper part) hook-curves calculated from hybridizations of the HG-U95 (left) and DG-1 (right) Gene Chips (see also Figure 1). The dotted lines indicate the hybridization ranges characterized by predominantly non-specific (N) and specific (S) binding, by a mixture of significant S- and N-contributions (mix), by the progressive saturation of the probe spots with bound transcripts (sat) and by almost completely saturated probes (as). Affinity correction considerably changes the shape of the hook-curve and the extent of the hybridization ranges. The corrected hook-curve and the fit are characterized by their geometrical dimensions; width (β), height (~α), start- (Σ(0), Δ(0)) and end- (Σ(∞)) positions; which in turn characterize the particular hybridization in terms of the mean non-specific background contribution, the PM/MM-gain etc. (see Table 2 for details). Compare also with Figure 1: The HG-U95 data were taken from different experiment series (Affymetrix spiked-in series here [3] and Genelogic dilution series [1] in Figure 1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2543012&req=5

Figure 2: Hybridization ranges of the raw (lower part) and the corrected (upper part) hook-curves calculated from hybridizations of the HG-U95 (left) and DG-1 (right) Gene Chips (see also Figure 1). The dotted lines indicate the hybridization ranges characterized by predominantly non-specific (N) and specific (S) binding, by a mixture of significant S- and N-contributions (mix), by the progressive saturation of the probe spots with bound transcripts (sat) and by almost completely saturated probes (as). Affinity correction considerably changes the shape of the hook-curve and the extent of the hybridization ranges. The corrected hook-curve and the fit are characterized by their geometrical dimensions; width (β), height (~α), start- (Σ(0), Δ(0)) and end- (Σ(∞)) positions; which in turn characterize the particular hybridization in terms of the mean non-specific background contribution, the PM/MM-gain etc. (see Table 2 for details). Compare also with Figure 1: The HG-U95 data were taken from different experiment series (Affymetrix spiked-in series here [3] and Genelogic dilution series [1] in Figure 1).
Mentions: Figure 1 depicts a typical graphical output-summary of the hook-analysis for two hybridizations performed on two different chip-types taken from the Genelogic dilution [1] and the GoldenSpike [2] experimental series (see also Figure 2 with data taken from the HG-U95 Latin square spiked-in series [3]). The Δ-vs-Σ plots characterize the hybridization of the particular chip. They are obtained by transforming the probe intensities of one GeneChip microarray into Δ = logIPM - logIMM and Σ = 0.5(logIPM + logIMM) coordinates and subsequent smoothing (IPM and IMM denote the spot intensities of the PM and MM probes after optical background correction; the logs are base 10 throughout the paper). The corrected version of the Δ-vs-Σ plot uses intensity values which are corrected for sequence-specific sensitivity effects. These plots are called hook-curves because of their typical shape. Additional characteristics of a particular chip-hybridization are the signal-density distribution and the four positional-dependent sensitivity profiles of the PM and MM probes upon specific and non-specific hybridization, respectively. These profiles are calculated from the intensity data of the chosen chip and used to correct the intensities for sequence-specific affinities.

Bottom Line: We show that the proper judgement of these effects requires the disentanglement of non-specific and specific hybridization which, otherwise, can lead to misinterpretations of expression changes.The consequences of modifying probe/target interactions by either changing the labelling protocol or by substituting RNA by DNA targets are demonstrated.The single-chip based hook-method provides accurate expression estimates and chip-summary characteristics using the natural metrics given by the hybridization reaction with the potency to develop new standards for microarray quality control and calibration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Interdisciplinary Centre for Bioinformatics, University of Leipzig, D-04107 Leipzig, Germany. binder@izbi.uni-leipzig.de

ABSTRACT

Background: Microarray experiments rely on several critical steps that may introduce biases and uncertainty in downstream analyses. These steps include mRNA sample extraction, amplification and labelling, hybridization, and scanning causing chip-specific systematic variations on the raw intensity level. Also the chosen array-type and the up-to-dateness of the genomic information probed on the chip affect the quality of the expression measures. In the accompanying publication we presented theory and algorithm of the so-called hook method which aims at correcting expression data for systematic biases using a series of new chip characteristics.

Results: In this publication we summarize the essential chip characteristics provided by this method, analyze special benchmark experiments to estimate transcript related expression measures and illustrate the potency of the method to detect and to quantify the quality of a particular hybridization. It is shown that our single-chip approach provides expression measures responding linearly on changes of the transcript concentration over three orders of magnitude. In addition, the method calculates a detection call judging the relation between the signal and the detection limit of the particular measurement. The performance of the method in the context of different chip generations and probe set assignments is illustrated. The hook method characterizes the RNA-quality in terms of the 3'/5'-amplification bias and the sample-specific calling rate. We show that the proper judgement of these effects requires the disentanglement of non-specific and specific hybridization which, otherwise, can lead to misinterpretations of expression changes. The consequences of modifying probe/target interactions by either changing the labelling protocol or by substituting RNA by DNA targets are demonstrated.

Conclusion: The single-chip based hook-method provides accurate expression estimates and chip-summary characteristics using the natural metrics given by the hybridization reaction with the potency to develop new standards for microarray quality control and calibration.

No MeSH data available.


Related in: MedlinePlus