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Statistical issues in the analysis of Illumina data.
Mark J Dunning1, Nuno L Barbosa-Morais, Andy G Lynch
1Department of Oncology, University of Cambridge, CRUK Cambridge Research Institute, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK. md392@cam.ac.uk
BMC Bioinformatics
|February 8, 2008
Summary
Pre-processing raw Illumina data is crucial for accurate gene expression analysis. Optimizing background subtraction and avoiding proprietary normalization methods improves differential expression detection and overall data reliability.
Area of Science:
- Genomics
- Bioinformatics
- Molecular Biology
Background:
- Illumina bead-based arrays are widely used for their high replication and data quality.
- Pre-processing of raw Illumina data has received limited attention.
- This study analyzes raw data from an Illumina spike-in experiment.
Purpose of the Study:
- Provide guidelines for analyzing Illumina expression data.
- Offer insights for developing new methodologies for Illumina technology.
- Evaluate the impact of pre-processing steps on data quality and analysis.
Main Methods:
- Analysis of raw Illumina spike-in experiment data.
- Comparison of Illumina's proprietary summary and background normalization methods.
- Application of variance-based weighting for differential expression analysis.
Main Results:
- Subtracting local background improves differential expression (DE) detection.
- Illumina's summary method effectively removes outliers.
- Proprietary background normalization can negatively impact DE analysis.
- Raw data quality assessment can reveal spatial artifacts missed in summarized data.
- Probe composition differences introduce variability not currently addressed.
Conclusions:
- Illumina's BeadStudio offers reasonable analysis but can be improved by omitting background normalization.
- Accessing raw data enables more thorough quality assessment and flexible analysis.
- Established tools can be used for gene expression studies on an appropriate scale.
- Similar improvements are anticipated for other Illumina assays.
