Open Access Highly Accessed Research article

Comparison of normalization methods for Illumina BeadChip HumanHT-12 v3

Ramona Schmid1, Patrick Baum1, Carina Ittrich1, Katrin Fundel-Clemens1, Wolfgang Huber2, Benedikt Brors3, Roland Eils34, Andreas Weith1, Detlev Mennerich15 and Karsten Quast1*

Author Affiliations

1 Boehringer Ingelheim Pharma GmbH & Co. KG, Birkendorfer Str. 65, 88397, Biberach/Riss, Germany

2 EMBL-EBI, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK

3 Theoretical Bioinformatics Department, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany

4 Department of Bioinformatics and Functional Genomics, Institute of Pharmacy and Molecular Biotechnology (IPMB) and BioQuant, University of Heidelberg, 69120 Heidelberg, Germany

5 Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, CT 06877, USA

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BMC Genomics 2010, 11:349  doi:10.1186/1471-2164-11-349

Published: 2 June 2010

Additional files

Additional file 1:

-log10(p-values) against MSQbetween where MSQbetween ≤ 5. MSQs were calculated based on the gene expression measured for the three sample groups analyzed, namely untreated HaCaT cells after 2, 4, and 12 hours. Results obtained for the different pre-processing methods used are displayed. The blue line represents a loess-curve fitted to the values.

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Additional file 2:

Density plots of MSQwithin (blue) and MSQbetween (red). MSQs were calculated based on the gene expression measured for the three sample groups analyzed, namely untreated HaCaT cells after 2, 4, and 12 hours. Results obtained for the different pre-processing methods used are displayed. The grey dashed line indicates the expected value for the MSQbetween of 1.33 based on 6, 6, and 7 as measurements for the group means of four replicates for three time points.

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Additional file 3:

Volcano plots. Log2 ratios and p-values for the comparison of untreated HaCaT cells at 4 hours compared to 2 hours, 12 hours compared to 2 hours, and 12 hours compared to 4 hours were calculated based on the gene expression measured. Displayed are the -log10(p-value) against log2 ratio for the respective comparisons and the different normalization methods used. The blue line represents a loess-curve fitted to the values.

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Additional file 4:

Residual standard deviation against minimum expression intensity. For each pre-processing method, standard deviation of the residuals observed for the regression fitted to the expression intensities are plotted against minimum expression intensity of each probe. The blue line represents a loess-curve fitted to the values.

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Additional file 5:

Residual standard deviation against mean expression intensity. For each pre-processing method, standard deviation of the residuals observed for the regression fitted to the expression intensities are plotted against mean expression intensity of each probe. The blue line represents a loess-curve fitted to the values.

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Additional file 6:

Scatterplots between replicates. After application of different normalization methods, expression values for the replicates are plotted against each other. The orange line indicates the main diagonal.

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Additional file 7:

Ranking of AUC values. AUC values as calculated for the pseudo-ROC analysis displayed in Figure 9 are ranked and cut-offs for the three bins are chosen based on the jumps visible at 0.86 and 0.89.

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Additional file 8:

Results of qRT-PCR. 2-ΔΔCt [37] values represent the observed fold changes between HaCaT cells stimulated with TGF-β (UT+TGFβ) and unstimulated cells (UT) at the three different time points measured.

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Additional file 9:

Orthogonal regression between qRT-PCR and normalization based log2 ratios. Regression of log2 ratios based on different normalization methods (y-axis) against qRT-PCR log2 ratios (x-axis). Equations and the respective regression lines are displayed in red. The grey dashed line indicates the main diagonal.

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