<i>Xenopus tropicalis</i> Genome Re-Scaffolding and Re-Annotation Reach the Resolution Required for <i>In Vivo</i> ChIA-PET Analysis

<div><p>Genome-wide functional analyses require high-resolution genome assembly and annotation. We applied ChIA-PET to analyze gene regulatory networks, including 3D chromosome interactions, underlying thyroid hormone (TH) signaling in the frog <i>Xenopus tropicalis</i>. As the available versions of <i>Xenopus tropicalis</i> assembly and annotation lacked the resolution required for ChIA-PET we improve the genome assembly version 4.1 and annotations using data derived from the paired end tag (PET) sequencing technologies and approaches (e.g., DNA-PET [gPET], RNA-PET etc.). The large insert (~10Kb, ~17Kb) paired end DNA-PET with high throughput NGS sequencing not only significantly improved genome assembly quality, but also strongly reduced genome “fragmentation”, reducing total scaffold numbers by ~60%. Next, RNA-PET technology, designed and developed for the detection of full-length transcripts and fusion mRNA in whole transcriptome studies (ENCODE consortia), was applied to capture the 5' and 3' ends of transcripts. These amendments in assembly and annotation were essential prerequisites for the ChIA-PET analysis of TH transcription regulation. Their application revealed complex regulatory configurations of target genes and the structures of the regulatory networks underlying physiological responses. Our work allowed us to improve the quality of <i>Xenopus tropicalis</i> genomic resources, reaching the standard required for ChIA-PET analysis of transcriptional networks. We consider that the workflow proposed offers useful conceptual and methodological guidance and can readily be applied to other non-conventional models that have low-resolution genome data.</p></div

RNA-PET efficiently captures transcripts ends.

<p>A. Overlap between RNA-Seq reads and Ensembl and RNA-PET-based models. B. Demarcation of gene model boundaries by RNA-PET. The histogram shows the relative size of Ensembl gene models in bins of various sizes. C. Enrichment of RNA-Pol II around Ensembl gene models and RNA-PET-based models. This shows that RNA-Pol II density fits well with RNA-PET based models, but not Ensembl models.</p

Examples of genome annotation improvements.

<p>Track order: Ensembl models, RNA-PET based models, RNA-PET ditags and RNA-Seq reads density. A, B, C: <i>sumo1</i>, <i>cadm2</i> and <i>kiaa1958</i> loci. D: Un-annotated gene split over scaffold_1031 and scaffold_1460.</p

Benefit of genome re-annotation with RNA-PET for ChIA-PET analysis.

<p>A. Genomic view of an un-annotated gene. Track order: Ensembl genes, RNA-PET-based models, ChIA-PET TR binding density, RNA Pol-II binding density, RNA-Seq reads density with (+T₃) and without (-T₃) THs treatment. B. Close up of TR binding sites. Track order: Ensembl genes, RNA-PET-based genes, location of ChIP-qPCR probes, RNA-PET PETs, TR binding density and RNA Pol-II binding density. C: ChIP-qPCR validation of TR binding at locations shown in B. Ab: antibody, T₃: 3’,5,3’ triiodothyronine treatment. D: Transcriptional induction assayed by RT-qPCR.</p

Benefit of genome re-annotation with RNA-PET for ChIA-PET analysis.

<p>A. Large genomic view of the <i>bcl6</i> locus. Track order: Ensembl genes, RNA-PET-based models, ChIA-PET TR binding density, interaction PETs, RNA Pol-II binding density, RNA-Seq reads density with (+T₃) and without (-T₃) treatment with thyroid hormones. B. Close up on TR binding sites. Track order: Ensembl genes, RNA-PET-based genes, location of ChIP-qPCR probes, RNA-PET ditags, TR binding density and RNA Pol-II binding density. C: ChIP-qPCR validation of TR binding at locations shown in B. Ab: Antibody, T₃: 3’,5,3’ triiodothyronine treatment. D: Induction of <i>trpg1</i>, <i>lpp</i> and <i>bcl6</i> genes transcription assayed by RT-qPCR. E: Three-dimensional model of the locus topology.</p

Detection of Chromosomal Breakpoints in Patients with Developmental Delay and Speech Disorders

<div><p>Delineating candidate genes at the chromosomal breakpoint regions in the apparently balanced chromosome rearrangements (ABCR) has been shown to be more effective with the emergence of next-generation sequencing (NGS) technologies. We employed a large-insert (7–11 kb) paired-end tag sequencing technology (DNA-PET) to systematically analyze genome of four patients harbouring cytogenetically defined ABCR with neurodevelopmental symptoms, including developmental delay (DD) and speech disorders. We characterized structural variants (SVs) specific to each individual, including those matching the chromosomal breakpoints. Refinement of these regions by Sanger sequencing resulted in the identification of five disrupted genes in three individuals: guanine nucleotide binding protein, q polypeptide <i>(GNAQ),</i> RNA-binding protein, fox-1 homolog <i>(RBFOX3),</i> unc-5 homolog D (<i>C.elegans) (UNC5D</i>), transmembrane protein 47 (<i>TMEM47</i>), and X-linked inhibitor of apoptosis (<i>XIAP</i>). Among them, <i>XIAP</i> is the causative gene for the immunodeficiency phenotype seen in the patient. The remaining genes displayed specific expression in the fetal brain and have known biologically relevant functions in brain development, suggesting putative candidate genes for neurodevelopmental phenotypes. This study demonstrates the application of NGS technologies in mapping individual gene disruptions in ABCR as a resource for deciphering candidate genes in human neurodevelopmental disorders (NDDs).</p></div

SV identification based on the mapping pattern of dPET clusters.

<p>The dark red and pink arrows represent the 5′ and 3′ anchor regions of the dPET cluster, respectively. Black, white and blue horizontal lines represent chromosome segments. The red track represents the coverage of cPETs. The dotted lines indicate the connections between the two dPET clusters. The sub-types of insertions are as follows: (1) Intra-chromosomal direct forward insertion. (2) Intra-chromosomal direct backward insertion. (3) Intra-chromosomal inverted forward insertion. (4) Intra-chromosomal inverted backward insertion. (5) Deletion plus intra-chromosomal direct forward insertion. (6) Deletion plus intra-chromosomal inverted forward insertion. (7) Inter-chromosomal direct insertion. (8) Inter chromosome inverted insertion.</p

DNA-PET library construction, sequencing and mapping.

<p>(A) The genomic DNA was randomly sheared to different size range. (B) The very narrow region DNA fragments were obtained after size selection. (C) The purified DNA fragments were circularized, <i>EcoP15I</i> digested, sequencing adaptor ligated, and finally sequenced by SOLiD sequencer. (D) PET mapping span distribution of 1 kb (blue), 10 kb (red) and 20 kb (green) libraries. Based on the mapping pattern, PETs can be distinguished as concordant PETs and discordant PETs.</p

Reconstruction of the <i>BCR-ABL1</i> amplicon of K562.

<p>(A) Concordant tag distributions representing copy number are shown for amplified genomic regions (top, green track). Genomic segments between predicted breakpoints are indicated by colored arrows and dPET clusters with cluster sizes greater than 35 of predicted somatic rearrangements are represented by horizontal lines flanked by dark red and pink arrows indicating 5′ and 3′ anchor regions (middle). Small to large dPET clusters are arranged from top to bottom. Cluster sizes are indicated. High dPET cluster size of the CML causing <i>BCR-ABL1</i> translocation suggests that the rearrangement occurred early and that it has subsequently been amplified. Fusion points I–III correspond to panels C–D. (B) Fluorescence <i>in situ</i> hybridization (FISH) of <i>BCR-ABL1</i> rearrangement (fusion point I with cluster size 692). Yellow spots represent fusion signals and illustrate the amplification of <i>BCR-ABL1</i>. (C) FISH analysis of metaphase chromosomes of three high copy fusion points: I) probes used in B show fusion signals on two marker chromosomes and on chromosome 2q and normal localization on both rearranged chromosomes 9 and normal chromosome 22; the fusion on chromosome 2 has not been identified by DNA-PET most likely due to low sequence complexity at the break point or complex rearrangements, II) probes spanning the fusion point II (cluster size 259) show fusion signals on the same marker chromosomes and normal localization on both normal and rearranged chromosomes 9 and 13, III) probes spanning fusion point III (cluster size 218) show fusion signals on the same marker chromosomes and normal localization on both normal chromosome 22 and rearranged chromosomes 9. (D) Contigs (indicated by boxes) which were covered by PET mapping were concatenated by fusion-point-guided-concatenation method. The length of a contig is represented by the length of the box. Because of the size difference between chromosomes 1, 3, 9, 13, and 22, the length of chromosome 22 is represented by the length of contig/10,000 while the lengths of chromosomes 1, 3, 9, and 13 are represented by the length of contig/100,000. Any value less than 0.1 is rounded to 0.1; any value larger than 6 is rounded to 6. The thickness of borders of each contig represents the coverage (copy number). Red dashed edges represent dPET edges, while black bold edges represent cPET edges. The thickness of dPET edges represents the size of the corresponding dPET cluster. cPET edges have uniform thickness. Arrow heads pointing towards a contig indicate connections with the lower coordinates, arrow heads pointing away from a contig indicate connections with the higher coordinates.</p

Statistics of PET sequencing.

1)<p>non-redundant.</p>2)<p>physical coverage.</p>3)<p>concordant PET.</p>4)<p>proportion of cPET to total NR PET.</p>5)<p>discordant PET.</p>6)<p>proportion of dPET to total NR PET.</p>7)<p>number of dPET involved in the dPET clusters with cluster count ≥3.</p