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Microarray data mining using Bioconductor packages
Haisheng Nie1, Pieter BT Neerincx2, Jan van der Poel1, Francesco Ferrari3,
Silvio Bicciato4, Jack AM Leunissen2 and Martien AM Groenen*1
Address: 1Animal Breeding and Genomics Centre, Wageningen University, Marijkeweg 40, P.O. Box 338, 6700 AH, Wageningen, The Netherlands,
2Laboratory of Bioinformatics, Wageningen University, Dreijenlaan 3, P.O. Box 569, 6700 AN, Wageningen, The Netherlands, 3Department of
Biology, University of Padova, Via G. Colombo 3, 35121, Padova, Italy and 4Department of Biomedical Sciences, University of Modena and Reggio
Emilia, via Campi 287, 41100, Modena, Italy
Email: Haisheng Nie - haisheng.nie@wur.nl; Pieter BT Neerincx - pieter.neerincx@gmail.com; Jan van der Poel - jan.vanderpoel@wur.nl;
Francesco Ferrari - francesco.ferrari@unipd.it; Silvio Bicciato - silvio.bicciato@unimore.it; Jack AM Leunissen - jack.leunissen@wur.nl;
Martien AM Groenen* - martien.groenen@wur.nl
* Corresponding author
Abstract
Background: This paper describes the results of a Gene Ontology (GO) term enrichment analysis
of chicken microarray data using the Bioconductor packages. By checking the enriched GO terms
in three contrasts, MM8-PM8, MM8-MA8, and MM8-MM24, of the provided microarray data during
this workshop, this analysis aimed to investigate the host reactions in chickens occurring shortly
after a secondary challenge with either a homologous or heterologous species of Eimeria. The
results of GO enrichment analysis using GO terms annotated to chicken genes and GO terms
annotated to chicken-human orthologous genes were also compared. Furthermore, a locally
adaptive statistical procedure (LAP) was performed to test differentially expressed chromosomal
regions, rather than individual genes, in the chicken genome after Eimeria challenge.
Results: GO enrichment analysis identified significant (raw p-value < 0.05) GO terms for all three
contrasts included in the analysis. Some of the GO terms linked to, generally, primary immune
responses or secondary immune responses indicating the GO enrichment analysis is a useful
approach to analyze microarray data. The comparisons of GO enrichment results using chicken
gene information and chicken-human orthologous gene information showed more refined GO
terms related to immune responses when using chicken-human orthologous gene information, this
suggests that using chicken-human orthologous gene information has higher power to detect
significant GO terms with more refined functionality. Furthermore, three chromosome regions
were identified to be significantly up-regulated in contrast MM8-PM8 (q-value < 0.01).
Conclusion: Overall, this paper describes a practical approach to analyze microarray data in farm
animals where the genome information is still incomplete. For farm animals, such as chicken, with
currently limited gene annotation, borrowing gene annotation information from orthologous genes
from EADGENE and SABRE Post-analyses Workshop
Lelystad, The Netherlands. 12–14 November 2008
Published: 16 July 2009
BMC Proceedings 2009, 3(Suppl 4):S9 doi:10.1186/1753-6561-3-S4-S9
<supplement> <title> <p>EADGENE and SABRE Post-analyses Workshop</p> </title> <editor>Dirk-Jan de Koning</editor> <sponsor> <note>The publication of these proceedings was supported by the EC-funded Network of Excellence EADGENE (EC contract number FOOD-CT-2004-506416).</note> </sponsor> <note>Proceedings</note> <url>http://www.biomedcentral.com/content/pdf/1753-6561-3-S4-info.pdf</url> </supplement>
This article is available from: http://www.biomedcentral.com/1753-6561/3/S4/S9
© 2009 Nie et al; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 5
(page number not for citation purposes)
in well-annotated species, such as human, will help improve the pathway analysis results

BMC Proceedings 2009, 3(Suppl 4):S9 http://www.biomedcentral.com/1753-6561/3/S4/S9
immune response at 8 hours after homologous MM chal-
lenge.
These results clearly show the induction of different
immune responses (primary vs. secondary) in chicken
that encountered an Eimeria infection for the first time
and chicken that had gone through an Eimeria infection at
an earlier time in their life.
(2) MM8-MA8 contrast
No major differences on immune response related GO
terms were identified in the MM8-MA8 contrast, these
results show that, heterologous challenge MA triggers a
very similar immune response as MM. Interestingly, the
genes up-regulated in the MM8-MA8 contrast show an
enrichment of GO term like "cell death" and "apoptosis",
suggesting that the heterologous challenge caused more
severe lesions in the chickens as compared to an homolo-
gous challenge.
No evidence shows that MM8 and MA8 trigger different
immune responses in chicken, although the enriched GO
terms indicate a more severe pathogenesis in case of het-
erologous challenge.
(3) MM8-MM24 contrast
As described in the MM8-PM8 contrast result, the homol-
ogous challenge already triggered a secondary immune
response at 8 hours. No significant GO terms related to
secondary immune response were found in MM8-MM24
contrast. The up-regulated genes in MM8-MM24 have
enriched GO terms like "positive regulation of NF-kappaB
transcription factor activity", and the down-regulated
genes in MM8-MM24 have enriched GO terms like, "T cell
receptor signalling pathway" and "interleukin-2 produc-
tion". NF-kappaB is a key regulator of several important
immune-related pathways, this suggests that immune
response activators were already highly up-regulated at 8
hours compared to 24 hours and that a secondary
immune responses kept on increasing from 8 hours to 24
hours after homologous challenge with MM.
Multiple testing problem
We have applied "BH" FDR control method for correction
for multiple testing using R package multtest [12] and
found only a few significant GO terms after correction
(data not shown). In this analysis we used threshold of
raw p-value < 0.05, the major reasons of not using the
FDR control methods are (a) the structure of the GO
graph is in conflict with the assumption of independence
for the test and (b) multiple testing correction methods do
not change the overall ranks of the results, using raw p-
value at cut-off would still identify the relative important
Annotation Sources comparison
In this section, GO enrichment analysis results using
chicken gene annotation and chicken-human ortholo-
gous gene annotation are compared. All the GO term
enrichment analysis results of this comparison are availa-
ble in the Additional file 2 and Additional file 3. The over-
lap of the results of the GO term enrichment analysis
using the chicken gene information and using the
chicken-human orthologous gene information is shown
in Figure 1. The overlap of the significant GO terms iden-
tified by both annotation sources is limited. Enriched GO
terms using chicken genes and using chicken-human
orthologous genes, as described above, gave a reasonably
good insight of the underlying biological processes in this
the experiment. The enriched GO terms based on the
chicken annotation directly didn't reveal much detail in
the ongoing processes after either homologous challenge
or heterologous challenge (see Additional file 2). The
enriched GO term using the chicken-human orthologous
gene information had a higher power to detect significant
GO terms (see Additional file 3), which can be explained
by the higher coverage of annotation (GO terms) using
this approach.
Performing the GO enrichment analysis using chicken-
human orthologous genes, on one hand, extensively
increased the coverage of the gene annotation of this
chicken oligo array platform. Consequently, this increases
the power of the hypergeometric test by having more
annotated genes in the DE gene lists. On the other hand,
care has to be taken by using this approach, as human and
chicken are evolutionarily far apart. Therefore, some of
Comparison of GO term enrichment analysis resultsFigure 1
Comparison of GO term enrichment analysis results.
Overlap of significantly enriched GO terms (raw p-value <
0.05) between the uses of chicken gene information versus



 










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GO terms in the results. chicken-human orthologous gene information.

BMC Proceedings 2009, 3(Suppl 4):S9 http://www.biomedcentral.com/1753-6561/3/S4/S9
the chicken-specific immune response processes may not
be identified using this approach. Nevertheless, this
approach helps researchers working with farm animals,
e.g. chicken, to increase the biological insight from their
microarray data by using human orthologous gene infor-
mation.
Differentially expressed chromosomal regions
Instead of testing enrichment of GO terms, chromosomal
locations could be used as "annotation" to test whether
certain chromosomal locations are more actively
expressed than other regions. In this analysis, the differen-
tially expressed chromosomal locations were identified
using locally adaptive procedure (LAP). In total, three sig-
nificant regions were up-regulated and one region was
down-regulated comparing PM and MM infections (see
details of those regions in Figure 2 and Additional file 4).
No significant regions were identified in other contrasts.
The identified differentially expressed chromosomal
regions indicate that some of the co-localized genes are
co-regulated during homologous challenge by MM, this
region-wide gene expression regulation mechanism was
reported in several other species [13,14].
Conclusion
The GO term enrichment analysis provided a good insight
in the biological processes involved in the Eimeria infec-
tion experiments. The GO enrichment analysis using sev-
eral bioconductor packages described in this paper
provides a practical, yet powerful, way of analyzing micro-
array data. Furthermore, the results suggest that using
chicken-human orthologous gene information provides
better insight in the biological processes underlying this
specific microarray experiment than by using the annota-
tion of chicken genes alone. This approach will be a help-
ful general method for researchers working with
microarray data in species with less well annotated-
genomes, like those of farm animals. Furthermore, LAP
analysis approach is a relatively new and very useful way
to be applied in microarray analysis to identify differen-
tially expressed chromosomal regions under specific
experimental conditions.
List of abbreviations used
DE: Differentially Expressed; FDR: False Discovery Rate;
GO: Gene Ontology; GO_BP: Gene Ontology Biological
Process; PM: PBS-E. Maxima; MM: E. maxima-E. Maxima;
Differentially expressed chromosomal regions for contrast MM8-PM8Figu 2
Differentially expressed chromosomal regions for contrast MM8-PM8. This figure showed the differentially
expressed chromosomal regions for MM8.PM8 contrast (q-value < 0.01). In total three regions were up-regulated and one
region was down-regulated. Red showed the up-regulated chromosomal regions, and Green showed the down-regulated Page 4 of 5
(page number not for citation purposes)
regions.

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BMC Proceedings 2009, 3(Suppl 4):S9 http://www.biomedcentral.com/1753-6561/3/S4/S9
MA: E. maxima-E. acervulina; LAP: locally adaptive statisti-
cal procedure
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
HN analyzed the data and drafted the manuscript, all
other authors helped to improve the manuscript. PBTN
and JAML helped with the re-annotation of the array, JP
and MAMG contributed to the biological interpretation of
the results, FF and SB performed the analysis of differen-
tially expressed chromosomal regions. All authors read
and approved the final manuscript.
Additional material
Acknowledgements
This work was funded by the EADGENE network. The authors wish to
acknowledge Caroline Channing and the other organizers for organizing
oligo array, Dr. Annemarie Rebel and colleagues from Animal Sciences
Group in Lelystad for providing the microarray data from the chicken infec-
tion experiment.
This article has been published as part of BMC Proceedings Volume 3 Sup-
plement 4, 2009: EADGENE and SABRE Post-analyses Workshop. The full
contents of the supplement are available online at http://www.biomedcen
tral.com/1753-6561/3?issue=S4.
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Additional file 1
GO enrichment analysis results with selected immune related GO
terms. This table shows GO enrichment results with selected GO_BP
terms. (For contrasts MM8.PM8 and MM8.MM24 results, only
immune-related GO_BP terms which have at least two genes linked to
each one of them were included).
Click here for file
[http://www.biomedcentral.com/content/supplementary/1753-
6561-3-S4-S9-S1.xls]
Additional file 2
GO term enrichment results (raw p-value <0.05) using chicken genes.
This table shows the GO enrichment analysis results using chicken gene
information.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1753-
6561-3-S4-S9-S2.xls]
Additional file 3
GO term enrichment results (raw p-value < 0.05) using chicken-
human orthologous genes. This table shows the GO term enrichment
analysis results using chicken-human orthologous genes information.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1753-
6561-3-S4-S9-S3.xls]
Additional file 4
Differentially expressed chromosomal regions for MM8-PM8 contrast.
This table shows the chromosomal locations of three up-regulated chromo-
somal regions and one down-regulated chromosomal region for MM8-
PM8 contrast.
Click here for file
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6561-3-S4-S9-S4.xls]yours — you keep the copyright
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the workshop, Christophe Klopp and Pierrot Casel from Institut National
de la Recherche Agrinomique (INRA) for re-annotating the chicken 20 k