Iain Milne

TOPALi v2

Summary: TOPALi v2 simplifies and automates the use of several methods for the evolutionary analysis of multiple sequence alignments. Jobs are submitted from a Java graphical user interface as TOPALi web services to either run remotely on high-performance computing clusters or locally (with multiple cores supported). Methods available include model selection and phylogenetic tree estimation using the Bayesian inference and maximum likelihood (ML) approaches, in addition to recombination detection methods. The optimal substitution model can be selected for protein or nucleic acid (standard, or protein-coding using a codon position model) data using accurate statistical criteria derived from ML co-estimation of the tree and the substitution model. Phylogenetic software available includes PhyML, RAxML and MrBayes. Availability: Freely downloadable from http://www.topali.org for Windows, Mac OS X, Linux and Solaris. Contact: iain.milne@scri.ac.uk

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.

TOPALi: software for automatic identification of recombinant sequences within DNA multiple alignments

SUMMARY TOPALi is a new Java graphical analysis application that allows the user to identify recombinant sequences within a DNA multiple alignment (either automatically or via manual investigation). TOPALi allows a choice of three statistical methods to predict the positions of breakpoints due to past recombination. The breakpoint predictions are then used to identify putative recombinant sequences and their relationships to other sequences. In addition to its sophisticated interface, TOPALi can import many sequence formats, estimate and display phylogenetic trees and allow interactive analysis and/or automatic HTML report generation. AVAILABILITY TOPALi is freely available from http://www.bioss.ac.uk/software.html

Difficulties in Learning and Teaching Programming—Views of Students and Tutors

We have conducted a web-based questionnaire on the various concepts and topics of object-oriented programming that students on introductory courses found most difficult to cope with.A statistical analysis of our results shows that those topics that rely on a clear understanding of pointers and memory-related concepts (such as copy constructors and virtual functions) prove to be the most difficult. In other words, we believe these concepts are only hard because of the students inability to comprehend what is happening to their program in memory, as they are incapable of creating a clear mental model of its execution.These results would suggest that a clearer approach to teaching these topics would be beneficial to students. We are currently working on a visualization-based approach to address these issues.

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 .

Effects of ascertainment bias and marker number on estimations of barley diversity from high-throughput SNP genotype data

The capability of molecular markers to provide information of genetic structure is influenced by their number and the way they are chosen. This study evaluates the effects of single nucleotide polymorphism (SNP) number and selection strategy on estimates of germplasm diversity and population structure for different types of barley germplasm, namely cultivar and landrace. One hundred and sixty-nine barley landraces from Syria and Jordan and 171 European barley cultivars were genotyped with 1536 SNPs. Different subsets of 384 and 96 SNPs were selected from the 1536 set, based on their ability to detect diversity in landraces or cultivated barley in addition to corresponding randomly chosen subsets. All SNP sets except the landrace-optimised subsets underestimated the diversity present in the landrace germplasm, and all subsets of SNP gave similar estimates for cultivar germplasm. All marker subsets gave qualitatively similar estimates of the population structure in both germplasm sets, but the 96 SNP sets showed much lower data resolution values than the larger SNP sets. From these data we deduce that pre-selecting markers for their diversity in a germplasm set is very worthwhile in terms of the quality of data obtained. Second, we suggest that a properly chosen 384 SNP subset gives a good combination of power and economy for germplasm characterization, whereas the rather modest gain from using 1536 SNPs does not justify the increased cost and 96 markers give unacceptably low performance. Lastly, we propose a specific 384 SNP subset as a standard genotyping tool for middle-eastern landrace barley.

An eQTL Analysis of Partial Resistance to Puccinia hordei in Barley

Background Genetic resistance to barley leaf rust caused by Puccinia hordei involves both R genes and quantitative trait loci. The R genes provide higher but less durable resistance than the quantitative trait loci. Consequently, exploring quantitative or partial resistance has become a favorable alternative for controlling disease. Four quantitative trait loci for partial resistance to leaf rust have been identified in the doubled haploid Steptoe (St)/Morex (Mx) mapping population. Further investigations are required to study the molecular mechanisms underpinning partial resistance and ultimately identify the causal genes. Methodology/Principal Findings We explored partial resistance to barley leaf rust using a genetical genomics approach. We recorded RNA transcript abundance corresponding to each probe on a 15K Agilent custom barley microarray in seedlings from St and Mx and 144 doubled haploid lines of the St/Mx population. A total of 1154 and 1037 genes were, respectively, identified as being P. hordei-responsive among the St and Mx and differentially expressed between P. hordei-infected St and Mx. Normalized ratios from 72 distant-pair hybridisations were used to map the genetic determinants of variation in transcript abundance by expression quantitative trait locus (eQTL) mapping generating 15685 eQTL from 9557 genes. Correlation analysis identified 128 genes that were correlated with resistance, of which 89 had eQTL co-locating with the phenotypic quantitative trait loci (pQTL). Transcript abundance in the parents and conservation of synteny with rice allowed us to prioritise six genes as candidates for Rphq11, the pQTL of largest effect, and highlight one, a phospholipid hydroperoxide glutathione peroxidase (HvPHGPx) for detailed analysis. Conclusions/Significance The eQTL approach yielded information that led to the identification of strong candidate genes underlying pQTL for resistance to leaf rust in barley and on the general pathogen response pathway. The dataset will facilitate a systems appraisal of this host-pathogen interaction and, potentially, for other traits measured in this population.

Exome sequencing of geographically diverse barley landraces and wild relatives gives insights into environmental adaptation

After domestication, during a process of widespread range extension, barley adapted to a broad spectrum of agricultural environments. To explore how the barley genome responded to the environmental challenges it encountered, we sequenced the exomes of a collection of 267 georeferenced landraces and wild accessions. A combination of genome-wide analyses showed that patterns of variation have been strongly shaped by geography and that variant-by-environment associations for individual genes are prominent in our data set. We observed significant correlations of days to heading (flowering) and height with seasonal temperature and dryness variables in common garden experiments, suggesting that these traits were major drivers of environmental adaptation in the sampled germplasm. A detailed analysis of known flowering-associated genes showed that many contain extensive sequence variation and that patterns of single- and multiple-gene haplotypes exhibit strong geographical structuring. This variation appears to have substantially contributed to range-wide ecogeographical adaptation, but many factors key to regional success remain unidentified.

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

Chromatin state analysis of the barley epigenome reveals a higher‐order structure defined by H3K27me1 and H3K27me3 abundance

Summary Combinations of histones carrying different covalent modifications are a major component of epigenetic variation. We have mapped nine modified histones in the barley seedling epigenome by chromatin immunoprecipitation next‐generation sequencing (ChIP‐seq). The chromosomal distributions of the modifications group them into four different classes, and members of a given class also tend to coincide at the local DNA level, suggesting that global distribution patterns reflect local epigenetic environments. We used this peak sharing to define 10 chromatin states representing local epigenetic environments in the barley genome. Five states map mainly to genes and five to intergenic regions. Two genic states involving H3K36me3 are preferentially associated with constitutive gene expression, while an H3K27me3‐containing genic state is associated with differentially expressed genes. The 10 states display striking distribution patterns that divide barley chromosomes into three distinct global environments. First, telomere‐proximal regions contain high densities of H3K27me3 covering both genes and intergenic DNA, together with very low levels of the repressive H3K27me1 modification. Flanking these are gene‐rich interior regions that are rich in active chromatin states and have greatly decreased levels of H3K27me3 and increasing amounts of H3K27me1 and H3K9me2. Lastly, H3K27me3‐depleted pericentromeric regions contain gene islands with active chromatin states separated by extensive retrotransposon‐rich regions that are associated with abundant H3K27me1 and H3K9me2 modifications. We propose an epigenomic framework for barley whereby intergenic H3K27me3 specifies facultative heterochromatin in the telomere‐proximal regions and H3K27me1 is diagnostic for constitutive heterochromatin elsewhere in the barley genome.