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Published on: October 3, 2025
Comparison of three microarray probe annotation pipelines: differences in strategies and their effect on downstream
Pieter Bt Neerincx1, Pierrot Casel2, Dennis Prickett3
1Laboratory of Bioinformatics, Wageningen University and Research centre (WUR), P.O. Box 569, 6700 AN Wageningen, The Netherlands.
Comparing three annotation pipelines (IMAD, OligoRAP, sigReannot) for microarray analysis revealed significant differences in gene linking and functional analysis results. Optimizing annotation strategies is crucial for reliable biological interpretation in experiments like those with Eimeria-infected chickens.
Area of Science:
- Genomics
- Bioinformatics
- Functional Genomics
Background:
- Accurate annotation linking oligonucleotide probes to target genes is critical for microarray-based functional analysis.
- The ARK-Genomics Chicken 20 K array annotation was updated using IMAD, OligoRAP, and sigReannot pipelines.
- This study compares the annotation strategies and outcomes of these pipelines.
Purpose of the Study:
- To compare the annotation strategies and results of IMAD, OligoRAP, and sigReannot pipelines.
- To analyze the impact of annotation differences on functional analysis, specifically in an Eimeria-infected chicken experiment.
- To propose guidelines for optimal annotation strategies in microarray analysis.
Main Methods:
- Utilized IMAD, OligoRAP, and sigReannot pipelines for updating oligonucleotide probe annotation.
- Assessed target specificity based on oligo-to-target gene alignments and filter thresholds.
- Compared gene linking outcomes and Gene Ontology (GO) term enrichment analysis results across pipelines.
Main Results:
- All three pipelines updated annotation and target specificity with varying resolution.
- Consensus gene linking was achieved for 44.0% of oligos, with 31.0% unassigned by any pipeline.
- Annotation differences significantly impacted GO term enrichment analysis, with only 67.2% consensus on enriched terms.
Conclusions:
- Annotation tools require flexible thresholds for target specificity and metadata on oligo-annotation relationships.
- Users need to balance reliability and coverage in annotation for accurate functional analysis.
- Discrepancies in annotation significantly affect functional analysis outcomes, as seen in GO term enrichment.
