Genomic targets of HOP2 are enriched for features found at recombination hotspots.

HOP2 is a conserved protein that plays a positive role in homologous chromosome pairing and a separable role in preventing illegitimate connections between nonhomologous chromosome regions during meiosis. We employed ChIP-seq to discover that Arabidopsis HOP2 binds along the length of all chromosomes, except for centromeric and nucleolar organizer regions, and no binding sites were detected in the organelle genomes. A large number of reads were assigned to the HOP2 locus itself, yet TAIL-PCR and SNP analysis of the aligned sequences indicate that many of these reads originate from the transforming T-DNA, supporting the role of HOP2 in preventing nonhomologous exchanges. The 292 ChIP-seq peaks are largely found in promoter regions and downstream from genes, paralleling the distribution of recombination hotspots, and motif analysis revealed that there are several conserved sequences that are also enriched at crossover sites. We conducted coimmunoprecipitation of HOP2 followed by LC-MS/MS and found enrichment for several proteins, including some histone variants and modifications that are also known to be associated with recombination hotspots. We propose that HOP2 may be directed to chromatin motifs near double strand breaks, where homology checks are proposed to occur.

BioViz Connect: Web Application Linking CyVerse Cloud Resources to Genomic Visualization in the Integrated Genome Browser.

Genomics researchers do better work when they can interactively explore and visualize data. Due to the vast size of experimental datasets, researchers are increasingly using powerful, cloud-based systems to process and analyze data. These remote systems, called science gateways, offer user-friendly, Web-based access to high performance computing and storage resources, but typically lack interactive visualization capability. In this paper, we present BioViz Connect, a middleware Web application that links CyVerse science gateway resources to the Integrated Genome Browser (IGB), a highly interactive native application implemented in Java that runs on the user's personal computer. Using BioViz Connect, users can 1) stream data from the CyVerse data store into IGB for visualization, 2) improve the IGB user experience for themselves and others by adding IGB specific metadata to CyVerse data files, including genome version and track appearance, and 3) run compute-intensive visual analytics functions on CyVerse infrastructure to create new datasets for visualization in IGB or other applications. To demonstrate how BioViz Connect facilitates interactive data visualization, we describe an example RNA-Seq data analysis investigating how heat and desiccation stresses affect gene expression in the model plant Arabidopsis thaliana. The RNA-Seq use case illustrates how interactive visualization with IGB can help a user identify problematic experimental samples, sanity-check results using a positive control, and create new data files for interactive visualization in IGB (or other tools) using a Docker image deployed to CyVerse via the Terrain API. Lastly, we discuss limitations of the technologies used and suggest opportunities for future work. BioViz Connect is available from https://bioviz.org.

Integrated Genome Browser App Store.

SUMMARY: Rapid progress in genome science requires equally rapid visualization software development so that researchers can better explore and understand novel datasets. To make developing new visualizations faster and easier, we previously re-factored the Integrated Genome Browser (IGB), a desktop Java application with dozens of features, into a pluggable application framework that can accept new functionality as plug-ins, called IGB Apps. However, developers lacked a centralized location for sharing Apps, making it hard to connect with potential users. To fill this gap, we created an App Store for IGB, a user-friendly Web site for developers to release and document Apps, and for users to find them. AVAILABILITY AND IMPLEMENTATION: The IGB App Store is available from https://bioviz.org.

Sp6/Epiprofin is a master regulator in the developing tooth.

Tooth development involves the coordinated transcriptional regulation of extracellular matrix proteins produced by ameloblasts and odontoblasts. In this study, whole-genome ChIP-seq analysis was applied to identify the transcriptional regulatory gene targets of Sp6 in mesenchymal cells of the developing tooth. Bioinformatic analysis of a pool of Sp6 target peaks identified the consensus nine nucleotide binding DNA motif CTg/aTAATTA. Consistent with these findings, a number of enamel and dentin matrix genes including amelogenin (Amelx), ameloblastin (Ambn), enamelin (Enam) and dental sialophosphoprotein (Dspp), were identified to contain Sp6 target sequences. Sp6 peaks were also found in other important tooth genes including transcription factors (Dlx2, Dlx3, Dlx4, Dlx5, Sp6, Sp7, Pitx2, and Msx2) and extracellular matrix-related proteins (Col1a2, Col11a2, Halpn1). Unsupervised UMAP clustering of tooth single cell RNA-seq data confirmed the presence of Sp6 transcripts co-expressed with many of the identified target genes within ameloblasts and odontoblasts. Lastly, transcriptional reporter assays using promoter fragments from the Hapln1 and Sp6 gene itself revealed that Sp6 co-expression enhanced gene transcriptional activity. Taken together these results highlight that Sp6 is a major regulator of multiple extracellular matrix genes in the developing tooth.

AINTEGUMENTA and AINTEGUMENTA-LIKE6 directly regulate floral homeotic, growth, and vascular development genes in young Arabidopsis flowers.

Arabidopsis flower primordia give rise to organ primordia in stereotypical positions within four concentric whorls. Floral organ primordia in each whorl undergo distinct developmental programs to become one of four organ types (sepals, petals, stamens, and carpels). The Arabidopsis transcription factors AINTEGUMENTA (ANT) and AINTEGUMENTA-LIKE6 (AIL6) are required for correct positioning of floral organ initiation, contribute to the specification of floral organ identity, and regulate the growth and morphogenesis of developing floral organs. To gain insight into the molecular mechanisms by which ANT and AIL6 contribute to floral organogenesis, we identified the genome-wide binding sites of both ANT and AIL6 in stage 3 flower primordia, the developmental stage at which sepal primordia become visible and class B and C floral homeotic genes are first expressed. AIL6 binds to a subset of ANT sites, suggesting that AIL6 regulates some but not all of the same target genes as ANT. ANT- and AIL6-binding sites are associated with genes involved in many biological processes related to meristem and flower organ development. Comparison of genes associated with both ANT and AIL6 ChIP-Seq peaks and those differentially expressed after perturbation of ANT and/or AIL6 activity identified likely direct targets of ANT and AIL6 regulation. These include class B and C floral homeotic genes, growth regulatory genes, and genes involved in vascular development.

Whole genome bisulfite sequencing of Medicago truncatula A17 wild type and lss mutants.

OBJECTIVES: Earlier work in our lab identified a spontaneous mutant (likesunnsupernodulator-lss) in Medicago truncatula, resulting in increased nodulation. Molecular genetic evidence indicated the phenotype was due to an unknown lesion resulting in cis-silencing of the SUNN gene. Altered methylation of the promoter was suspected, but analysis of the SUNN promoter by bisulfite sequencing at the time of publication revealed no significant methylation differences between the SUNN promoter in wild type and lss plants. Using advances in methylome generation we compared the methylome of wild type and the lss mutant in the larger 810 kB area of the genome where lss maps. DATA DESCRIPTION: The data show the distribution of types of methylation across the entire genome between A17 wild type and lss mutants, the number of differentially methylated cytosines between genotypes, and the overall pattern of gene methylation between genotypes. We expect the wild type data will be especially useful as a reference for other investigations of methylation using M. truncatula.

Discovery of genomic variations by whole-genome resequencing of the North American Araucana chicken.

Gallus gallus (chicken) is phenotypically diverse, with over 60 recognized breeds, among the myriad species within the Aves lineage. Domestic chickens have been under artificial selection by humans for thousands of years for agricultural purposes. The North American Araucana (NAA) breed arose as a cross between the Chilean "Collonocas" that laid blue eggs and was rumpless and the "Quetros" that had unusual tufts but with tail. NAAs were introduced from South America in the 1940s and have been kept as show birds by enthusiasts since then due to several distinctive traits: laying eggs with blue eggshells, characteristic ear-tufts, a pea comb, and rumplessness. The population has maintained variants for clean-faced and tufted, as well as tailed and rumplessness traits making it advantageous for genetic studies. Genome resequencing of six NAA chickens with a mixture of these traits was done to 71-fold coverage using Illumina HiSeq 2000 paired-end reads. Trimmed and concordant reads were mapped to the Gallus_gallus-5.0 reference genome (galGal5), generated from a female Red Junglefowl (UCD001). To identify candidate genes that are associated with traits of the NAA, their genome was compared with the Korean Araucana, Korean Domestic and White Leghorn breeds. Genomic regions with significantly reduced levels of heterogeneity were detected on five different chromosomes in NAA. The sequence data generated confirm the identity of variants responsible for the blue eggshells, pea comb, and rumplessness traits of NAA and propose one for ear-tufts.

An 'eFP-Seq Browser' for visualizing and exploring RNA sequencing data.

Improvements in next-generation sequencing technologies have resulted in dramatically reduced sequencing costs. This has led to an explosion of '-seq'-based methods, of which RNA sequencing (RNA-seq) for generating transcriptomic data is the most popular. By analysing global patterns of gene expression in organs/tissues/cells of interest or in response to chemical or environmental perturbations, researchers can better understand an organism's biology. Tools designed to work with large RNA-seq data sets enable analyses and visualizations to help generate hypotheses about a gene's function. We present here a user-friendly RNA-seq data exploration tool, called the 'eFP-Seq Browser', that shows the read map coverage of a gene of interest in each of the samples along with 'electronic fluorescent pictographic' (eFP) images that serve as visual representations of expression levels. The tool also summarizes the details of each RNA-seq experiment, providing links to archival databases and publications. It automatically computes the reads per kilobase per million reads mapped expression-level summaries and point biserial correlation scores to sort the samples based on a gene's expression level or by how dissimilar the read map profile is from a gene splice variant, to quickly identify samples with the strongest expression level or where alternative splicing might be occurring. Links to the Integrated Genome Browser desktop visualization tool allow researchers to visualize and explore the details of RNA-seq alignments summarized in eFP-Seq Browser as coverage graphs. We present four cases of use of the eFP-Seq Browser for ABI3, SR34, SR45a and U2AF65B, where we examine expression levels and identify alternative splicing. The URL for the browser is https://bar.utoronto.ca/eFP-Seq_Browser/. OPEN RESEARCH BADGES: This article has earned an Open Data Badge for making publicly available the digitally-shareable data necessary to reproduce the reported results. Tool is at https://bar.utoronto.ca/eFP-Seq_Browser/; RNA-seq data at https://s3.amazonaws.com/iplant-cdn/iplant/home/araport/rnaseq_bam/ and https://s3.amazonaws.com/iplant-cdn/iplant/home/araport/rnaseq_bam/Klepikova/. Code is available at https://github.com/BioAnalyticResource/eFP-Seq-Browser.

Many rice genes are differentially spliced between roots and shoots but cytokinin has minimal effect on splicing.

Alternatively spliced genes produce multiple spliced isoforms, called transcript variants. In differential alternative splicing, transcript variant abundance differs across sample types. Differential alternative splicing is common in animal systems and influences cellular development in many processes, but its extent and significance is not as well known in plants. To investigate differential alternative splicing in plants, we examined RNA-Seq data from rice seedlings. The data included three biological replicates per sample type, approximately 30 million sequence alignments per replicate, and four sample types: roots and shoots treated with exogenous cytokinin delivered hydroponically or a mock treatment. Cytokinin treatment triggered expression changes in thousands of genes but had negligible effect on splicing patterns. However, many genes were differentially spliced between mock-treated roots and shoots, indicating that our methods were sufficiently sensitive to detect differential splicing between data sets. Quantitative fragment analysis of reverse transcriptase-PCR products made from newly prepared rice samples confirmed 9 of 10 differential splicing events between rice roots and shoots. Differential alternative splicing typically changed the relative abundance of splice variants that co-occurred in a data set. Analysis of a similar (but less deeply sequenced) RNA-Seq data set from Arabidopsis showed the same pattern. In both the Arabidopsis and rice RNA-Seq data sets, most genes annotated as alternatively spliced had small minor variant frequencies. Of splicing choices with abundant support for minor forms, most alternative splicing events were located within the protein-coding sequence and maintained the annotated reading frame. A tool for visualizing protein annotations in the context of genomic sequence (ProtAnnot) together with a genome browser (Integrated Genome Browser) were used to visualize and assess effects of differential splicing on gene function. In general, differentially spliced regions coincided with conserved protein domains, indicating that differential alternative splicing is likely to affect protein function between root and shoot tissue in rice.

ProtAnnot: an App for Integrated Genome Browser to display how alternative splicing and transcription affect proteins.

UNLABELLED: One gene can produce multiple transcript variants encoding proteins with different functions. To facilitate visual analysis of transcript variants, we developed ProtAnnot, which shows protein annotations in the context of genomic sequence. ProtAnnot searches InterPro and displays profile matches (protein annotations) alongside gene models, exposing how alternative promoters, splicing and 3' end processing add, remove, or remodel functional motifs. To draw attention to these effects, ProtAnnot color-codes exons by frame and displays a cityscape graphic summarizing exonic sequence at each position. These techniques make visual analysis of alternative transcripts faster and more convenient for biologists. AVAILABILITY AND IMPLEMENTATION: ProtAnnot is a plug-in App for Integrated Genome Browser, an open source desktop genome browser available from http://www.bioviz.org CONTACT: aloraine@uncc.edu.

Integrated genome browser: visual analytics platform for genomics.

MOTIVATION: Genome browsers that support fast navigation through vast datasets and provide interactive visual analytics functions can help scientists achieve deeper insight into biological systems. Toward this end, we developed Integrated Genome Browser (IGB), a highly configurable, interactive and fast open source desktop genome browser. RESULTS: Here we describe multiple updates to IGB, including all-new capabilities to display and interact with data from high-throughput sequencing experiments. To demonstrate, we describe example visualizations and analyses of datasets from RNA-Seq, ChIP-Seq and bisulfite sequencing experiments. Understanding results from genome-scale experiments requires viewing the data in the context of reference genome annotations and other related datasets. To facilitate this, we enhanced IGB's ability to consume data from diverse sources, including Galaxy, Distributed Annotation and IGB-specific Quickload servers. To support future visualization needs as new genome-scale assays enter wide use, we transformed the IGB codebase into a modular, extensible platform for developers to create and deploy all-new visualizations of genomic data. AVAILABILITY AND IMPLEMENTATION: IGB is open source and is freely available from http://bioviz.org/igb CONTACT: aloraine@uncc.edu.

Analysis of pollen-specific alternative splicing in Arabidopsis thaliana via semi-quantitative PCR.

Alternative splicing enables a single gene to produce multiple mRNA isoforms by varying splice site selection. In animals, alternative splicing of mRNA isoforms between cell types is widespread and supports cellular differentiation. In plants, at least 20% of multi-exon genes are alternatively spliced, but the extent and significance of tissue-specific splicing is less well understood, partly because it is difficult to isolate cells of a single type. Pollen is a useful model system to study tissue-specific splicing in higher plants because pollen grains contain only two cell types and can be collected in large amounts without damaging cells. Previously, we identified pollen-specific splicing patterns by comparing RNA-Seq data from Arabidopsis pollen and leaves. Here, we used semi-quantitative PCR to validate pollen-specific splicing patterns among genes where RNA-Seq data analysis indicated splicing was most different between pollen and leaves. PCR testing confirmed eight of nine alternative splicing patterns, and results from the ninth were inconclusive. In four genes, alternative transcriptional start sites coincided with alternative splicing. This study highlights the value of the low-cost PCR assay as a method of validating RNA-Seq results.

A novel gain-of-function mutation of the proneural IRX1 and IRX2 genes disrupts axis elongation in the Araucana rumpless chicken.

Axis elongation of the vertebrate embryo involves the generation of cell lineages from posterior progenitor populations. We investigated the molecular mechanism governing axis elongation in vertebrates using the Araucana rumpless chicken. Araucana embryos exhibit a defect in axis elongation, failing to form the terminal somites and concomitant free caudal vertebrae, pygostyle, and associated tissues of the tail. Through whole genome sequencing of six Araucana we have identified a critical 130 kb region, containing two candidate causative SNPs. Both SNPs are proximal to the IRX1 and IRX2 genes, which are required for neural specification. We show that IRX1 and IRX2 are both misexpressed within the bipotential chordoneural hinge progenitor population of Araucana embryos. Expression analysis of BRA and TBX6, required for specification of mesoderm, shows that both are downregulated, whereas SOX2, required for neural patterning, is expressed in ectopic epithelial tissue. Finally, we show downregulation of genes required for the protection and maintenance of the tailbud progenitor population from the effects of retinoic acid. Our results support a model where the disruption in balance of mesoderm and neural fate results in early depletion of the progenitor population as excess neural tissue forms at the expense of mesoderm, leading to too few mesoderm cells to form the terminal somites. Together this cascade of events leads to axis truncation.

Genome-wide association mapping and identification of candidate genes for the rumpless and ear-tufted traits of the Araucana chicken.

Araucana chickens are known for their rounded, tailless rumps and tufted ears. Inheritance studies have shown that the rumpless (Rp) and ear-tufted (Et) loci each act in an autosomal dominant fashion, segregate independently, and are associated with an increased rate of embryonic mortality. To find genomic regions associated with Rp and Et, we generated genome-wide SNP profiles for a diverse population of 60 Araucana chickens using the 60 K chicken SNP BeadChip. Genome-wide association studies using 40 rumpless and 11 tailed birds showed a strong association with rumpless on Gga 2 (P(raw) = 2.45×10(-10), P(genome) = 0.00575), and analysis of genotypes revealed a 2.14 Mb haplotype shared by all rumpless birds. Within this haplotype, a 0.74 Mb critical interval containing two Iroquois homeobox genes, Irx1 and Irx2, was unique to rumpless Araucana chickens. Irx1 and Irx2 are central for developmental prepatterning, but neither gene is known to have a role in mechanisms leading to caudal development. A second genome-wide association analysis using 30 ear-tufted and 28 non-tufted birds revealed an association with tufted on Gga 15 (P(raw) = 6.61×10(-7), P(genome) = 0.0981). We identified a 0.58 Mb haplotype common to tufted birds and harboring 7 genes. Because homozygosity for Et is nearly 100% lethal, we employed a heterozygosity mapping approach to prioritize candidate gene selection. A 60 kb region heterozygous in all Araucana chickens contains the complete coding sequence for TBX1 and partial sequence for GNB1L. TBX1 is an important transcriptional regulator of embryonic development and a key genetic determinant of human DiGeorge syndrome. Herein, we describe localization of Rp and Et and identification of positional candidate genes.