de Jong S et al. BMC Genomics 2010, 11:20 http://www.biomedcentral.com/1471-2164/11/20 The same post-hoc analyses were performed to assess preservation of the modules of interest in networks con- structed using either amygdalar or hippocampus sam- ples (from both strains) with probes colored according to the initial network (constructed on all samples from both strains). Visual inspection of the hierarchical clus- tering trees of the amygdalar and hippocampus samples separately shows preservation of the Magenta module, but not of the Red or Pink modules (see Additional File 4). Magenta module eigengene significance remained high for strain differences in the networks created with hippocampal samples (r = 0.99, p = 1.1e-14) and amyg- dala (r = 0.99, p = 1.3e-14) separately. We found that the Magenta module was highly preserved between amygdalar and hippocampal networks (r = 0.80, p < 10e-03). Polymorphic SNPs in module genes and probes To investigate whether genetic variation between the two inbred strains could affect module detection, we collected SNP data from genes represented in the Magenta, Pink and Red modules (Table 1). SNPs were counted in target genes with a 10 kb region around the gene location. A comparison between the three modules showed that the Magenta module contained the smallest number of total SNPs relative to the number of interro- gated base pairs compared to the Pink (p < 2.2e-16) and Red (p < 2.2e-16) modules; the Pink module contained significantly more total SNPs than the Red module (p < 2.2e-16). However, the Magenta module, which was enriched with genes that are differentially expressed between A/J and C57BL/6J, contained significantly more non-synonymous coding SNPs than those found in the Pink (p = 2.6e-07) and Red (p = 3e-03) modules; the Pink and Red modules did not differ in their numbers of non-synonymous coding SNPs (p = 0.04). When checking probe sequences for known SNPs between A/J and C57BL/6J, the Magenta module was composed of significantly more SNP-containing probes (32 of 76 probes) than the Red module (3 out of 198 probes, p < 2.2e-16) and the Pink module (1 out of 84 probes, p = 1.3e-11). The number of probes containing SNPs did not differ significantly between the Red and Pink modules. In addition, sequencing of eleven of the top probes of the Magenta modules without known Page 6 of 14 SNP revealed that 91% (10 of the 11) contain a poly- morphism between C57BL/6J and A/J, with the C57BL/ 6J genomic sequence being identical to the probe sequence in all cases. Results are given in Additional File 5. In order to better understand the high frequency of sequence variation in probe regions of the Magenta module genes, we also investigated whether the target genes of the probes in the Magenta module with and without a SNP were cis- or trans-regulated genes. We consulted the publicly available WTCCC heterogeneous stock database containing expression quantitative trait loci (eQTL) in hippocampus for this purpose [36]. This data was also collected using the Illumina platform and differentiates between real cis-effects and possible false cis-acting eQTLs resulting from probes with and with- out a SNP in the sequence. Of the 32 target genes with probes resident in the Magenta module and containing a SNP, more possible false cis-acting eQTLs were detected in the WTCCC database than the 44 remaining probes of the Magenta module (13 out of 32 vs. 7 out of 44 probes, p = 0.02). Moreover, probes in the Magenta module without known SNPs were significantly enriched with real cis-acting eQTLs compared to the probe sequences containing a SNP (16 out of 44 vs. 3 out of 32, p < 0.01). Gene ontology enrichment analysis We used Ingenuity Pathways Analysis (Ingenuity® Sys- tems, http://www.ingenuity.com) to study the ontology of genes in the Pink and Red modules - these modules were enriched for genes that are differentially expressed between amygdala and hippocampus. For the Red mod- ule (165 out of 198 genes are known) the top-3 overre- presented subcategories within ‘diseases and disorders’ were Neurological Disease (p = 7.6e-7 - 3.3e-2), Genetic Disorder ((1.5e-6 - 2.7e-2) and Psychological Disorder (p = 1.2e-5 - 1.5e-2). The category ‘physiological system development and function’ yielded the following top-3: Behavior (p = 3.3e-6 - 2.2e-2), Nervous System Develop- ment and Function (p = 4.4e-5 - 3.3e-2) and Cardiovas- cular System Development and Function (p = 5.7e-5 - 2.9e-2). The Pink module (72 out of 84 genes were in the Ingenuity Pathways Knowledge Base) yielded no sig- nificant results using this threshold. The Magenta Table 1 SNP counts within target genes in significant modules Magenta Pink Red Total bp checked 21,218,367 8,637,477 15,909,861 Total SNPs 7,128 8,193 10,464 % of bp 0.03 0.09 0.07 Non-synonymous coding SNPs 59 19 49 % of SNPs found 0.83 0.23 0.47 The first column refers to the number of base pairs checked within a module. To include possible regulatory regions, a margin of 10 kb before and after each gene was used. The second column contains the total number of SNPs found, and the third column shows this number relative to the number of base pairs checked. Non-synonymous coding SNPs are represented in column four, and column five shows the percentage of non-synonymous coding SNPs compared to the total number of SNPs found.