Integrating multiple genome annotation databases improves the interpretation of microarray gene expression data

Abstract

BackgroundThe Affymetrix GeneChip is a widely used gene expression profiling platform. Since the chips were originally designed, the genome databases and gene definitions have been considerably updated. Thus, more accurate interpretation of microarray data requires parallel updating of the specificity of GeneChip probes. We propose a new probe remapping protocol, using the zebrafish GeneChips as an example, by removing nonspecific probes, and grouping the probes into transcript level probe sets using an integrated zebrafish genome annotation. This genome annotation is based on combining transcript information from multiple databases. This new remapping protocol, especially the new genome annotation, is shown here to be an important factor in improving the interpretation of gene expression microarray data.ResultsTranscript data from the RefSeq, GenBank and Ensembl databases were downloaded from the UCSC genome browser, and integrated to generate a combined zebrafish genome annotation. Affymetrix probes were filtered and remapped according to the new annotation. The influence of transcript collection and gene definition methods was tested using two microarray data sets. Compared to remapping using a single database, this new remapping protocol results in up to 20% more probes being retained in the remapping, leading to approximately 1,000 more genes being detected. The differentially expressed gene lists are consequently increased by up to 30%. We are also able to detect up to three times more alternative splicing events. A small number of the bioinformatics predictions were confirmed using real-time PCR validation.ConclusionsBy combining gene definitions from multiple databases, it is possible to greatly increase the numbers of genes and splice variants that can be detected in microarray gene expression experiments.

Long-term IL-33–producing epithelial progenitor cells in chronic obstructive lung disease

“…Differential expression was assessed using linear models and empirical Bayes moderated F statistics, as implemented in the LIMMA package (85). To improve interpretation (86), bead types were annotated using annotations from Illumina (87), ReMOAT (88), AILUN (89), AceView (90), and Ensembl (91). Differences in gene expression were considered significant if P < 0.05 after adjustment for multiple testing (92), limiting the false discovery rate to <5%.…”

Inhibition of the Pim1 Oncogene Results in Diminished Visual Function

“…Embryos were obtained by natural spawning and developmental stages established by time and morphological criteria. Microarray experiments were performed as previously described [11]. Eyes were dissected from 3, 4 and 5 days post fertilization (dpf) zebrafish larvae.…”

“…Gene annotation was based on the zebrafish genome version 9 (Zv9) and integrating gene transcript collections from multiple genome annotation databases [11]. Transcript data from the RefSeq, GenBank and Ensembl databases were downloaded from the UCSC genome browser [12].…”

“…Transcripts were clustered into genes from overlapping coding exons. A customized probe remapping was performed as previously described [11]. In order to take advantage of the human genome annotation, human-zebrafish homology data were downloaded from Ensembl [13], BioMart [14], ZFIN [15], and NCBI HomoloGene [16].…”

“…The threshold for differentially expressed genes was set as adjusted p-value <0.05 and fold change ≥1.5 or ≤0.67. For genes with multiple probe sets, a revised Splicing Index is calculated [11]. If the Splicing Index is ≤1 and ≥−1, the probe set expressions were averaged to calculate gene level expression.…”