Additional file 2: Figure S1.

Sequence analysis of new KIR haplotypes. Cartoons illustrating the gene content structures of recombinant haplotypes aligned with their putative parent haplotypes are shown with DNA sources and haplotype names respectively labeled. The extent of fosmid clones and long-range PCR products sequenced are indicated with green bars aligned with each set of cartoons. A color-coded key at the bottom indicates different types of gene rearrangements found in the new haplotypes. Figure S2. Phylogenetic and breakpoint analysis suggest possible recombination points in the centromeric and telomeric regions. (A) The 2DL23-2DS35 intergenic region and the exon 1-5 regions of 2DL2, 2DL3, and 2DS2 were subjected to phylogenetic analysis as described in Methods. Analysis of the 2DL23-2DS35 intergenic region revealed the presence of 4 major linkage groups and analysis of 2DL2, 2DL3, and 2DS2 sequences revealed 3 linkage groups, indicated by colored shading and labels. (B) Breakpoint analysis of three regions is presented. The pairwise identity plots indicate where crossover events are most likely to have occurred. Keys at the bottom of each plot identify the sequences that were used in the comparisons. Vertical bars at the top of each plot designate the positions of sequence differences. The exon-intron structures of the gene regions are indicated beneath each graph where appropriate with blue or pink designating the origins of the hybrid sequence. (C) Phylogenetic analysis was performed on two segments of KIR-3DL1 as indicated. The results for both regions are shown with the presence of the 2 major groups in blue (3DL1*015-like) or pink (3DL1*005-like). (D) Phylogenetic analysis was performed on three separate regions in KIR-3DL2 as indicated. Major groups of alleles are presented in green (3DL2*00701/*018), blue (3DL1*015-like) or pink (3DL1*005-like). Sequences and linkage information used in this analysis were from this study and previous haplotype sequencing [9]. Figure S3. (A) Identification of possible recombination events in the telomeric regions generating hybrid genes. Sequence alignments of 3DS1, 3DL1, and 3DL2 and the hybrid genes 3DS1/L1 and 3DL1/L2. In the sequence alignment plot, vertical bars represent SNP positions, color shading identifies the source gene, and the consensus gene structure is indicated beneath. Gene sequences are color-coded (3DS1, purple; 3DL1, blue; 3DL2 pink). We used full genomic sequence of 3DL1/L2 hybrid gene from EU267269. The orange shading represents a possible exchange segment between intron 5 and exon 6, flanked by L1PMA2 and MLT1D-like elements. Pairwise identity plots indicate where recombination events likely occurred. Keys at the bottom of each plot indicate the sequences that were used in the comparisons. Vertical bars at the top of each plot represent sequence differences. Haplotype motifs and exon-intron structures of 3DS1/3DL1 and 3DL1/3DL2 hybrid genes are shown. Color shading indicates the source content of the hybrid gene, aligned beneath the tA01-like motif within which they were identified. (B) Analysis of hybrid genes associated with KIR-2DL1, 2DS1, and 2DP1. Sequence alignment and breakpoint analysis comparing the genomic regions of 2DL1 (A/B), 2DS1, 2DP1 (A/B), and the hybrid genes 2DP1/L1 and 2DL1/S1. In the scale at the top, the green shading (darker) indicates the extent of sequence homology among the aligned sequences. A scaled cartoon of the consensus gene structure with repeat elements labeled is beneath the alignments. Two possible recombination sites in the intron 3 region indicated by red bars in the sequence alignment are supported by the sequence alignment and the pairwise identity plots. The patterns of mismatches (black hatches) indicate the positions of the minor allele SNP among the aligned sequences and above the pairwise identity graphs. Immediately beneath the graphs are color-coded keys identifying the sequences being analyzed. Breakpoint and phylogenetic analysis of the 2DL1A, 2DL1B, and 2DS1 sequences. The pairwise identity plot compares the sequences as indicated in the key, SNPs are represented by vertical bars above the graph, and the gene exon-intron structure is represented with boxes and lines at the bottom. Phylogenetic analysis distinguished three groups as indicated by color shading. Modeling of recombination between 2DL1, 2DS1, and 2DP1 in the intron 3 region. Consequent known hybrid genes are depicted as are hybrid genes not yet identified (boxed). (C) Putative recombination between the 2DL5 and 3DP1 genes is localized to the intron 2 regions. Sequence alignments of the genomic segment from exon 1 to intron 2 in KIR2DL5 and KIR3DP1 are shown. Source DNAs in the alignment are identified on the left and the structures of the recombinant segments are drawn beneath the alignment. The color-coded key at the bottom of the alignment indicates the origin of respective sequences. Breakpoint analysis revealed a likely recombination site in the intron 2 region. Pairwise identity plots of the 2DL5A, 2DL5B, and 3DP1 sequences are shown. The patterns of mismatches (black hatches) indicate the positions of the minor allele SNP among the aligned sequences and coloring indicating the sequence origin according to the key. KIR2DL5 was divided into two regions (exon 1 to exon 2 and intron 2 to exon 9) by phylogenetic analysis. Analysis of the exon 1-exon 2 region revealed two groups, 2DL5A (telomeric; blue) and 2DL5B (centromeric; pink). Analysis of the second region, intron 3 to exon 9, also revealed two groups, 2DL5-2DS3 (peach) and 2DL5-2DS5 (green). Figure S4. Putative recombination between the 2DL5 and 3DP1 genes is localized to the intron 2 regions. (A) Sequence alignments of the genomic segment from exon 1 to intron 2 in KIR2DL5 and KIR3DP1 are shown. Source DNAs in the alignment are identified on the left and the structures of the recombinant segments are drawn beneath the alignment. The color-coded key at the bottom of the alignment indicates the origin of respective sequences. (B) Breakpoint analysis revealed a likely recombination site in the intron 2 region. Pairwise identity plots of the 2DL5A, 2DL5B, and 3DP1 sequences are shown. The patterns of mismatches (black hatches) indicate the positions of the minor allele SNP among the aligned sequences and coloring indicating the sequence origin according to the key. (C) KIR2DL5 was divided into two regions (exon 1 to exon 2 and intron 2 to exon 9) by phylogenetic analysis. Analysis of the exon 1-exon 2 region revealed two groups, 2DL5A (telomeric; blue) and 2DL5B (centromeric; pink). Analysis of the second region, intron 3 to exon 9, also revealed two groups, 2DL5-2DS3 (peach) and 2DL5-2DS5 (green). Figure S5. Analysis of hybrid genes associated with KIR-2DL1, 2DS1, and 2DP1. (A) Sequence alignment and breakpoint analysis comparing the genomic regions of 2DL1 (cA01 motif/cB01 motif), 2DS1, 2DP1 (cA01 motif/cB01 motif), and the hybrid genes 2DP1/L1 and 2DL1/S1. In the scale at the top, the green shading (darker) indicates the extent of sequence homology among the aligned sequences. A scaled cartoon of the consensus gene structure with repeat elements labeled is beneath the alignments. Two possible recombination sites in the intron 3 region (red bars) are supported by the sequence alignment and the pairwise identity plots. The patterns of mismatches (black hatches) indicate the positions of the minor allele SNP among the aligned sequences and above the pairwise identity graphs. Immediately beneath the graphs are color-coded keys identifying the sequences being analyzed. (B) Breakpoint and phylogenetic analysis of the 2DL1A, 2DL1B, and 2DS1 sequences. The pairwise identity plot compares the sequences as indicated in the key, SNPs are represented by vertical bars above the graph, and the gene exon-intron structure is represented with boxes and lines at the bottom. Phylogenetic analysis distinguished three groups as indicated by color shading. (C) Modeling of recombination between 2DL1, 2DS1, and 2DP1 in the intron 3 region. Boxed genes are hybrid genes not yet identified. Figure S6. Peak ratio determines copy number for KIR haplotype analysis. Examples of ABI chromatograms are presented showing peak ratio differences from sequence analysis of assay 2DS1-4-002. This assay amplifies homologous sequences from three different loci as described in methods, with a key base distinguishing each locus at position 257. The five examples show different copy number ratios for 2DS1 (A):2DL1 (C):2DS4 (T) as indicated beneath each chromatogram. The copy number was validated from control samples where the complete phased genomic sequences from both haplotypes were available. (PDF 2241 kb)

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Pyo et al. BMC Genomics 2013 14:89   doi:10.1186/1471-2164-14-89