Paul D. Shaw

Using Tablet for visual exploration of second-generation sequencing data

The advent of second-generation sequencing (2GS) has provided a range of significant new challenges for the visualization of sequence assemblies. These include the large volume of data being generated, short-read lengths and different data types and data formats associated with the diversity of new sequencing technologies. This article illustrates how Tablet-a high-performance graphical viewer for visualization of 2GS assemblies and read mappings-plays an important role in the analysis of these data. We present Tablet, and through a selection of use cases, demonstrate its value in quality assurance and scientific discovery, through features such as whole-reference coverage overviews, variant highlighting, paired-end read mark-up, GFF3-based feature tracks and protein translations. We discuss the computing and visualization techniques utilized to provide a rich and responsive graphical environment that enables users to view a range of file formats with ease. Tablet installers can be freely downloaded from http://bioinf.hutton.ac.uk/tablet in 32 or 64-bit versions for Windows, OS X, Linux or Solaris. For further details on the Tablet, contact tablet@hutton.ac.uk.

Natural variation in a homolog of Antirrhinum CENTRORADIALIS contributed to spring growth habit and environmental adaptation in cultivated barley

As early farming spread from the Fertile Crescent in the Near East around 10,000 years before the present, domesticated crops encountered considerable ecological and environmental change. Spring-sown crops that flowered without the need for an extended period of cold to promote flowering and day length–insensitive crops able to exploit the longer, cooler days of higher latitudes emerged and became established. To investigate the genetic consequences of adaptation to these new environments, we identified signatures of divergent selection in the highly differentiated modern-day spring and winter barleys. In one genetically divergent region, we identify a natural variant of the barley homolog of Antirrhinum CENTRORADIALIS (HvCEN) as a contributor to successful environmental adaptation. The distribution of HvCEN alleles in a large collection of wild and landrace accessions indicates that this involved selection and enrichment of preexisting genetic variants rather than the acquisition of mutations after domestication.

Flapjack--graphical genotype visualization.

SUMMARY New software tools for graphical genotyping are required that can routinely handle the large data volumes generated by the high-throughput single-nucleotide polymorphism (SNP) platforms, genotyping-by-sequencing and other comparable genotyping technologies. Flapjack has been developed to facilitate analysis of these data, providing real time rendering with rapid navigation and comparisons between lines, markers and chromosomes, with visualization, sorting and querying based on associated data, such as phenotypes, quantitative trait loci or other mappable features. AVAILABILITY Flapjack is freely available for Microsoft Windows, Mac OS X, Linux and Solaris, and can be downloaded from http://bioinf.scri.ac.uk/flapjack .

Genome-wide association mapping of agronomic and morphologic traits in highly structured populations of barley cultivars.

Genome-wide association study (GWAS) has become an obvious general approach for studying traits of agricultural importance in higher plants, especially crops. Here, we present a GWAS of 32 morphologic and 10 agronomic traits in a collection of 615 barley cultivars genotyped by genome-wide polymorphisms from a recently developed barley oligonucleotide pool assay. Strong population structure effect related to mixed sampling based on seasonal growth habit and ear row number is present in this barley collection. Comparison of seven statistical approaches in a genome-wide scan for significant associations with or without correction for confounding by population structure, revealed that in reducing false positive rates while maintaining statistical power, a mixed linear model solution outperforms genomic control, structured association, stepwise regression control and principal components adjustment. The present study reports significant associations for sixteen morphologic and nine agronomic traits and demonstrates the power and feasibility of applying GWAS to explore complex traits in highly structured plant samples.

Plant snoRNA database

The Plant snoRNA database (http://www.scri.sari.ac.uk/plant_snoRNA/) provides information on small nucleolar RNAs from Arabidopsis and eighteen other plant species. Information includes sequences, expression data, methylation and pseudouridylation target modification sites, initial gene organization (polycistronic, single gene and intronic) and the number of gene variants. The Arabidopsis information is divided into box C/D and box H/ACA snoRNAs, and within each of these groups, by target sites in rRNA, snRNA or unknown. Alignments of orthologous genes and gene variants from different plant species are available for many snoRNA genes. Plant snoRNA genes have been given a standard nomenclature, designed wherever possible, to provide a consistent identity with yeast and human orthologues.

Arabidopsis nucleolar protein database (AtNoPDB)

The Arabidopsis Nucleolar Protein Database (http://bioinf.scri.sari.ac.uk/cgi-bin/atnopdb/home) provides information on 217 proteins identified in a proteomic analysis of nucleoli isolated from Arabidopsis cell culture. The database is organized on the basis of the Arabidopsis gene identifier number. The information provided includes protein description, protein class, whether or not the plant protein has a homologue in the most recent human nucleolar proteome and the results of reciprocal BLAST analysis of the human proteome. In addition, for one-third of the 217 Arabidopsis nucleolar proteins, localization images are available from analysis of full-length cDNA–green fluorescent protein (GFP) fusions and the strength of signal in different parts of the cell—nucleolus, nucleolus-associated structures, nucleoplasm, nuclear bodies and extra-nuclear—is provided. For each protein, the most likely human and yeast orthologues, where identifiable through BLASTX analysis, are given with links to relevant information sources.

An hnRNP-like RNA-binding protein affects alternative splicing by in vivo interaction with transcripts in Arabidopsis thaliana

Alternative splicing (AS) of pre-mRNAs is an important regulatory mechanism shaping the transcriptome. In plants, only few RNA-binding proteins are known to affect AS. Here, we show that the glycine-rich RNA-binding protein AtGRP7 influences AS in Arabidopsis thaliana. Using a high-resolution RT–PCR-based AS panel, we found significant changes in the ratios of AS isoforms for 59 of 288 analyzed AS events upon ectopic AtGRP7 expression. In particular, AtGRP7 affected the choice of alternative 5′ splice sites preferentially. About half of the events are also influenced by the paralog AtGRP8, indicating that AtGRP7 and AtGRP8 share a network of downstream targets. For 10 events, the AS patterns were altered in opposite directions in plants with elevated AtGRP7 level or lacking AtGRP7. Importantly, RNA immunoprecipitation from plant extracts showed that several transcripts are bound by AtGRP7 in vivo and indeed represent direct targets. Furthermore, the effect of AtGRP7 on these AS events was abrogated by mutation of a single arginine that is required for its RNA-binding activity. This indicates that AtGRP7 impacts AS of these transcripts via direct interaction. As several of the AS events are also controlled by other splicing regulators, our data begin to provide insights into an AS network in Arabidopsis.

Development and mapping of DArT markers within the Festuca - Lolium complex.

BackgroundGrasses are among the most important and widely cultivated plants on Earth. They provide high quality fodder for livestock, are used for turf and amenity purposes, and play a fundamental role in environment protection. Among cultivated grasses, species within the Festuca-Lolium complex predominate, especially in temperate regions. To facilitate high-throughput genome profiling and genetic mapping within the complex, we have developed a Diversity Arrays Technology (DArT) array for five grass species: F. pratensis, F. arundinacea, F. glaucescens, L. perenne and L. multiflorum.ResultsThe DArTFest array contains 7680 probes derived from methyl-filtered genomic representations. In a first marker discovery experiment performed on 40 genotypes from each species (with the exception of F. glaucescens for which only 7 genotypes were used), we identified 3884 polymorphic markers. The number of DArT markers identified in every single genotype varied from 821 to 1852. To test the usefulness of DArTFest array for physical mapping, DArT markers were assigned to each of the seven chromosomes of F. pratensis using single chromosome substitution lines while recombinants of F. pratensis chromosome 3 were used to allocate the markers to seven chromosome bins.ConclusionThe resources developed in this project will facilitate the development of genetic maps in Festuca and Lolium, the analysis on genetic diversity, and the monitoring of the genomic constitution of the Festuca × Lolium hybrids. They will also enable marker-assisted selection for multiple traits or for specific genome regions.

Small changes in ambient temperature affect alternative splicing in Arabidopsis thaliana

Alternative splicing (AS) gives rise to multiple mRNA isoforms from the same gene, providing possibilities to regulate gene expression beyond the level of transcription. In a recent paper in Nucleic Acids Research we used a high resolution RT-PCR based panel to study changes in AS patterns in plants with altered levels of an hnRNP-like RNA-binding protein in Arabidopsis thaliana. Furthermore, we detected significant changes in AS patterns between different Arabidopsis ecotypes. Here we investigated how small changes in ambient temperature affect AS. We found significant changes in AS for 12 of 28 investigated events (43%) upon transfer of Arabidopsis plants from 20°C to 16°C and for 6 of the 28 investigated events (21%) upon transfer from 20°C to 24°C.

Comparative visualization of genetic and physical maps with Strudel

Summary: Data visualization can play a key role in comparative genomics, for example, underpinning the investigation of conserved synteny patterns. Strudel is a desktop application that allows users to easily compare both genetic and physical maps interactively and efficiently. It can handle large datasets from several genomes simultaneously, and allows all-by-all comparisons between these. Availability and implementation: Installers for Strudel are available for Windows, Linux, Solaris and Mac OS X at http://bioinf.scri.ac.uk/strudel/. Contact: strudel@scri.ac.uk; micha.bayer@scri.ac.uk