title: Comparison of three microarray probe annotation pipelines: differences in strategies and their effect on downstream analysis.
abstract: BACKGROUND
  Reliable annotation linking oligonucleotide probes to target genes is essential for functional biological analysis of microarray experiments. We used the IMAD, OligoRAP and sigReannot pipelines to update the annotation for the ARK-Genomics Chicken 20 K array as part of a joined EADGENE/SABRE workshop. In this manuscript we compare their annotation strategies and results. Furthermore, we analyse the effect of differences in updated annotation on functional analysis for an experiment involving Eimeria infected chickens and finally we propose guidelines for optimal annotation strategies.
  
  
  RESULTS
  IMAD, OligoRAP and sigReannot update both annotation and estimated target specificity. The 3 pipelines can assign oligos to target specificity categories although with varying degrees of resolution. Target specificity is judged based on the amount and type of oligo versus target-gene alignments (hits), which are determined by filter thresholds that users can adjust based on their experimental conditions. Linking oligos to annotation on the other hand is based on rigid rules, which differ between pipelines.For 52.7% of the oligos from a subset selected for in depth comparison all pipelines linked to one or more Ensembl genes with consensus on 44.0%. In 31.0% of the cases none of the pipelines could assign an Ensembl gene to an oligo and for the remaining 16.3% the coverage differed between pipelines. Differences in updated annotation were mainly due to different thresholds for hybridisation potential filtering of oligo versus target-gene alignments and different policies for expanding annotation using indirect links. The differences in updated annotation packages had a significant effect on GO term enrichment analysis with consensus on only 67.2% of the enriched terms.
  
  
  CONCLUSIONS
  In addition to flexible thresholds to determine target specificity, annotation tools should provide metadata describing the relationships between oligos and the annotation assigned to them. These relationships can then be used to judge the varying degrees of reliability allowing users to fine-tune the balance between reliability and coverage. This is important as it can have a significant effect on functional microarray analysis as exemplified by the lack of consensus on almost one third of the terms found with GO term enrichment analysis based on updated IMAD, OligoRAP or sigReannot annotation.
authors.affiliation: Laboratory of Bioinformatics, Wageningen University and Research centre (WUR), P.O. Box 569, 6700 AN Wageningen, The Netherlands.
authors.forename: Pieter Bt
authors.initials: PB
authors.lastname: Neerincx
authors.affiliation: Sigenae UR875 Biométrie et Intelligence Artificielle/Génétique Cellulaire, Institut National de la Recherche Agrinomique (INRA), BP 52627, 31326 Castanet-Tolosan Cedex, France.
authors.forename: Pierrot
authors.initials: P
authors.lastname: Casel
authors.affiliation: Institute for Animal Health (IAH), Compton, nr Newbury, RG20 7NN, UK.
authors.forename: Dennis
authors.initials: D
authors.lastname: Prickett
authors.affiliation: Animal Breeding and Genomics Centre, Wageningen University and Research centre (WUR), P.O. Box 338, 6700 AH, Wageningen, The Netherlands.
authors.forename: Haisheng
authors.initials: H
authors.lastname: Nie
authors.affiliation: Institute for Animal Health (IAH), Compton, nr Newbury, RG20 7NN, UK.
authors.forename: Michael
authors.initials: M
authors.lastname: Watson
authors.affiliation: Laboratory of Bioinformatics, Wageningen University and Research centre (WUR), P.O. Box 569, 6700 AN Wageningen, The Netherlands.
authors.forename: Jack Am
authors.initials: JA
authors.lastname: Leunissen
authors.affiliation: Animal Breeding and Genomics Centre, Wageningen University and Research centre (WUR), P.O. Box 338, 6700 AH, Wageningen, The Netherlands.
authors.forename: Martien Am
authors.initials: MA
authors.lastname: Groenen
authors.affiliation: Sigenae UR875 Biométrie et Intelligence Artificielle/Génétique Cellulaire, Institut National de la Recherche Agrinomique (INRA), BP 52627, 31326 Castanet-Tolosan Cedex, France.
authors.forename: Christophe
authors.initials: C
authors.lastname: Klopp