Oren M et al. (2016), Short tandem repeats, segmental duplications, g...

ECB-ART-45010

BMC Genomics 2016 Nov 09;171:900. doi: 10.1186/s12864-016-3241-x.

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Short tandem repeats, segmental duplications, gene deletion, and genomic instability in a rapidly diversified immune gene family.

Oren M , Barela Hudgell MA , D'Allura B , Agronin J , Gross A , Podini D , Smith LC .

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BACKGROUND: Genomic regions with repetitive sequences are considered unstable and prone to swift DNA diversification processes. A highly diverse immune gene family of the sea urchin (Strongylocentrotus purpuratus), called Sp185/333, is composed of clustered genes with similar sequence as well as several types of repeats ranging in size from short tandem repeats (STRs) to large segmental duplications. This repetitive structure may have been the basis for the incorrect assembly of this gene family in the sea urchin genome sequence. Consequently, we have resolved the structure of the family and profiled the members by sequencing selected BAC clones using Illumina and PacBio approaches. RESULTS: BAC insert assemblies identified 15 predicted genes that are organized into three clusters. Two of the gene clusters have almost identical flanking regions, suggesting that they may be non-matching allelic clusters residing at the same genomic locus. GA STRs surround all genes and appear in large stretches at locations of putatively deleted genes. GAT STRs are positioned at the edges of segmental duplications that include a subset of the genes. The unique locations of the STRs suggest their involvement in gene deletions and segmental duplications. Genomic profiling of the Sp185/333 gene diversity in 10 sea urchins shows that no gene repertoires are shared among individuals indicating a very high gene diversification rate for this family. CONCLUSIONS: The repetitive genomic structure of the Sp185/333 family that includes STRs in strategic locations may serve as platform for a controlled mechanism which regulates the processes of gene recombination, gene conversion, duplication and deletion. The outcome is genomic instability and allelic mismatches, which may further drive the swift diversification of the Sp185/333 gene family that may improve the immune fitness of the species.

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Genes referenced: 185-333 LOC100887844 LOC100892350 LOC100893907 LOC115925415 LOC582503 LOC585204

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	Fig. 1. The Sp185/333 genes have two exons and a mosaic of elements in the second exon. a An alignment cartoon illustrates the structure of several genes with two exons (shown in relative size scale) and one intron (int; not shown to scale). Elements in the second exon are indicated as colored rectangles and gaps that have been artificially inserted to optimize the alignment are shown as horizontal black lines. All known elements are numbered at the top. Element patterns share mosaics of elements and naming of element patterns (on the left) are based on the sequence of element 10 (equivalent of element 15 in [25]). The imperfect, tandem type I repeats in the 5′ half of the second exon are indicated as red rectangles (elements 2–5) and have been evaluated computationally for duplications, deletions and recombinations [31]. Five additional types of repeats are imperfect and interspersed in elements 11–26 (see [20]). This figure is modified from the repeat-based alignment published in [20]. b The approximate locations of primers are indicated with arrows within the standard Sp185/333 gene structure. These primers are used to amplify Sp185/333 gene sequences and to identify the genes within the BAC insert assemblies. Primer sequences are listed in Additional file 1: Table S1. The arrows between (a) and (b) indicate the correlation between elements in (a) and locations of primers shown in (b)
	Fig. 2. Three distinct Sp185/333 gene amplicon size patterns are detected in large insert BAC clones. Amplicon peak height and area indicate three major amplicon size patterns from fragment analysis, which are designated as Cluster 1 (a), Cluster 2 (b) and Cluster 3 (c). Amplicon size patterns identified less often (1 or 2 clones of 27 analyzed; see Additional file 3: Figure S2A) and that partially match amplicons in either Cluster 1 or 2 are designated as Cluster 1′, Cluster 1″ (a) or Cluster 2′ (b). Fragment length analysis of each cluster was carried out for each of the corresponding BACs (listed below) using primers F6 and R9 (see Additional file 1: Table S1; Additional file 2: Figure S1B). Means and standard error were calculated when multiple BACs were used for analysis of Clusters 1 and 2. Cluster 1 BACs are 10A2, 10B1, 10C6, 4074 J14, 4079E24, 61 M13. Cluster 2 BACs are 4C3, 4007 J10, 4024 N22, 4029 F3, 4091I3, 10 K9, 64C18, 4011G4. Cluster 3 BACs are 10C18, 10H9, 10H10, 10 M18, 3090I9, 3104 N4, 3104P4, 4028 F7, 4067A10. Cluster 1′ BACs are 3033E12, 4093A10. Cluster 1″ BAC is 4093A10. The Cluster 2′ BAC is 409O3
	Fig. 3. Repetitive sequences in the Sp185/333 gene clusters are assembled correctly by PacBio but not by Illumina. Five BAC insert assemblies are compared to self by pairwise alignments and illustrated as dot plots. Lines and dots that are displaced from the central diagonal are repetitive sequences that include Sp185/333 gene sequences (regions with displaced diagonals are indicated with boxes) and STRs (dots). a The Sp185/333 gene clusters assembled from Illumina reads for BAC assemblies corresponding to Clusters 1, 2 and 3 have Sp185/333 genes that are fragmented and shorter than expected (the displaced diagonals are composed of short lines or dots) based on gene size predictions (not shown) and according to previous reports [20, 21]. b The Sp185/333 gene clusters assembled from PacBio reads for BACs compared to self. The PacBio assemblies show intact and longer displaced diagonals representing genes of expected length. The orientations of the genes are indicated by the displaced diagonals are either parallel (same orientation) or perpendicular (opposite orientation) to the central diagonal
	Fig. 4. Comparisons among BAC insert assemblies suggest two genomic loci for three Sp185/333 gene clusters. A map of all BAC insert sequences is based on pairwise alignments among all BAC insert sequences and indicates that Clusters 1 and 2 may be allelic. a Nine BAC insert sequences, including five that contain Sp185/333 gene Cluster 1 and four that contain Sp185/333 gene Cluster 2, match almost perfectly within the regions that flank the gene clusters. b The two BAC inserts that include Cluster 3 align separately as a different locus
	Fig. 5. Three clusters of Sp185/333 genes in the sequenced genome of S. purpuratus. The genes within Clusters 1, 2 and 3 range in size from 1170 to 1894 nt and are spaced apart by 3–12.8 kb. All genes have two exons, as indicated by the rectangle (first exon) and pentagon (second exon), which also indicates gene orientation within each cluster. Element patterns are listed above each gene and are indicated in different colors as initially defined by [21], which is identified by the shaded area in Cluster 1. All genes are surrounded by GA STRs (green triangles). The long stretches of GA STRs located on both sides of Cluster 3 are positioned at distances that correlate with corresponding genes in the other clusters. This map is based on three verified BAC assemblies (accession numbers: KU668452, KU668453, KU668454). Segmental duplications are surrounded by GAT STRs (black triangles denote GAT STRs of ≥ 35 repeats and gray triangles denote GAT STRs of 4–17 repeats; see Additional file 7: Table S3). Triangle location above or below the line indicates the orientation of the STRs, which is relative to the proximal gene
	Fig. 6. Tandem segmental duplications are bounded by GAT STRs. a Segmental duplications that include D1 genes are present in all three clusters (dark gray rectangles), are bounded by GAT STRs (black triangles denote GAT STRs of 35 or more repeats; gray triangles denote 4–17 repeats), and are indicated by the Cluster number and order of appearance (1–1, 1–2, etc.). 3* indicates a partial segmental duplication in Cluster 3, which is flanked by a GAT STR on the left and GA STR on to the right. The locations of putatively deleted genes in Cluster 3 are marked as “Gene?” in light gray that correlate with the positions of long GA repeats (see Fig. 5). b An alignment of the full length segmental duplications was employed for phylogenetic analysis by maximum parsimony using MEGA. Bootstrap numbers are indicated and based on 500 iterations. The regions surrounding B8 and B8a are defined as the outgroup (OG-1, OG-2). The sequences of the segmental duplications used for the alignment and phylogenetic tree are from BAC 10B1 (GenBank accession number KU668451) duplication 1–1 is nt 110752–115376; duplication 1–2 is nt 115460–120091; duplication 1–3 is nt 120177–124609; outgroup OG-1 is nt 106160–110660. BAC 10K9 (GenBank accession number KU668453) duplication 2–1 is nt 121690–126221; duplication 2–2 is nt 126365–130976; outgroup OG-2 is nt 117100–121600. BAC 3104P4 (GenBank accession number KU668454) duplication 3–1 is nt 91585–93433 and the partial duplication 3* is nt 96723–98626

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