RESEARCH our analysis set of I1 repeats, we mapped the ident- iﬁed intronic repeats from Satellog to the same version of the human genome reference using the exonic positions of the RefSeq-supported22 genes (curated non-redundant genes) retrieved by PipHelper (http://pipmaker.bx.psu.edu/cgi-bin/ piphelper/).27 The ﬁnal analysis dataset comprised I1 (TG/CA)n repeats within the 2.5 kilobase (kb) windows either downstream or upstream of neigh- bouring exons (Figure 1). We limited our analysis to those introns bigger than 2.5 kb. Chromosomal distribution of the genes containing I1 (TG/CA)n repeats Distribution of the genes containing I1 (TG/CA)n repeats across the human genome were tested against the chromosomal distribution of the human genome (version hg16), which has 18,299 RefSeq-supported22 genes. Over- or under- represented chromosomes were determined using binomial tests. A false discovery rate (FDR) of 5 per cent after the Benjamini – Hochberg (BH) cor- rection28 was used as the cut-off for statistical signiﬁcance. Gene ontology and pathway analyses We used the Database for Annotation, Visualization and Integrated Discovery (DAVID)29,30 (http://david. abcc.ncifcrf.gov) to identify enriched GO23 (http:// www.geneontology.org) or Protein ANalysis THrough Evolutionary Relationships (PANTHER)31 (http://www.pantherdb.org/pathway/) biological Zhang et al. processes, as well as known pathways such as those maintained in the KEGG24 (http://www.genome.jp/ kegg/), Biocarta (http://www.biocarta.com) and PANTHER31 databases among the genes containing long I1 (TG/CA)n repeats. Biological processes or pathways over-represented relative to the whole human genome (version hg16) were identiﬁed using Fisher’s exact test (ﬁve hits or more, FDR , 0.05 after BH correction28). Genotyping the I1 (TG/CA)n repeats To evaluate if the I1 (TG/CA)n repeats were poly- morphic, we genotyped a list of genes with relatively long (n 12) repeats in the 59 unrelated HapMap CEU samples. The gene expression levels of these samples previously had been measured using the Affymetrix GeneChipw Human Exon 1.0ST array (Affymetrix, Santa Clara, CA, USA) and are publicly available through the Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo/), acces- sion GSE7851.15 Ten genes with the top-ranking variability and associated with enriched pathways or biological processes were included in the genotyping (Table 1). DNA samples were extracted from LCLs purchased from the Coriell Institute for Medical Research (Camden, NJ, USA). Polymerase chain reaction (PCR), in combination with ﬂuorescently labelled oligonucleotide primers, was used to amplify the I1Rs containing (TG/CA)n repeats. Primers were designed to amplify the repeat region.32 One primer was labelled with 6-carboxyﬂuorescein at the 50 end. The primer sequences are listed in Table 1. Figure 1. Schematic of the intron 1 regions covered in the analysis. The 50 windows (2.5 kb) and 30 windows (2.5 kb) of the intron 1 regions were included in the analysis. (a) The intron 1 region is smaller than 5 kb. (b) The intron 1 region is larger than 5 kb. 238 # HENRY STEWART PUBLICATIONS 1479–7364. HUMAN GENOMICS. VOL 3. NO. 3. 236–245 APRIL 2009