RESUMEN
RNA-sequencing (RNA-seq) analysis of gene expression and alternative splicing should be routine and robust but is often a bottleneck for biologists because of different and complex analysis programs and reliance on specialized bioinformatics skills. We have developed the '3D RNA-seq' App, an R shiny App and web-based pipeline for the comprehensive analysis of RNA-seq data from any organism. It represents an easy-to-use, flexible and powerful tool for analysis of both gene and transcript-level gene expression to identify differential gene/transcript expression, differential alternative splicing and differential transcript usage (3D) as well as isoform switching from RNA-seq data. 3D RNA-seq integrates state-of-the-art differential expression analysis tools and adopts best practice for RNA-seq analysis. The program is designed to be run by biologists with minimal bioinformatics experience (or by bioinformaticians) allowing lab scientists to analyse their RNA-seq data. It achieves this by operating through a user-friendly graphical interface which automates the data flow through the programs in the pipeline. The comprehensive analysis performed by 3D RNA-seq is extremely rapid and accurate, can handle complex experimental designs, allows user setting of statistical parameters, visualizes the results through graphics and tables, and generates publication quality figures such as heat-maps, expression profiles and GO enrichment plots. The utility of 3D RNA-seq is illustrated by analysis of data from a time-series of cold-treated Arabidopsis plants and from dexamethasone-treated male and female mouse cortex and hypothalamus data identifying dexamethasone-induced sex- and brain region-specific differential gene expression and alternative splicing.
Asunto(s)
Empalme Alternativo , Arabidopsis/metabolismo , Corteza Cerebelosa/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Hipotálamo/metabolismo , RNA-Seq/métodos , ARN/genética , Animales , Arabidopsis/efectos de los fármacos , Corteza Cerebelosa/efectos de los fármacos , Frío , Biología Computacional/métodos , Dexametasona/farmacología , Glucocorticoides/farmacología , Hipotálamo/efectos de los fármacos , Ratones , ARN/metabolismo , Programas InformáticosRESUMEN
Undomesticated wild species, crop wild relatives, and landraces represent sources of variation for wheat improvement to address challenges from climate change and the growing human population. Here, we study 56,342 domesticated hexaploid, 18,946 domesticated tetraploid and 3,903 crop wild relatives in a massive-scale genotyping and diversity analysis. Using DArTseqTM technology, we identify more than 300,000 high-quality SNPs and SilicoDArT markers and align them to three reference maps: the IWGSC RefSeq v1.0 genome assembly, the durum wheat genome assembly (cv. Svevo), and the DArT genetic map. On average, 72% of the markers are uniquely placed on these maps and 50% are linked to genes. The analysis reveals landraces with unexplored diversity and genetic footprints defined by regions under selection. This provides fertile ground to develop wheat varieties of the future by exploring specific gene or chromosome regions and identifying germplasm conserving allelic diversity missing in current breeding programs.
Asunto(s)
Variación Genética , Genoma de Planta , Triticum/genética , Alelos , Domesticación , Genotipo , Modelos Genéticos , Polimorfismo de Nucleótido Simple , Alineación de Secuencia , TetraploidíaRESUMEN
BACKGROUND: The time required to analyse RNA-seq data varies considerably, due to discrete steps for computational assembly, quantification of gene expression and splicing analysis. Recent fast non-alignment tools such as Kallisto and Salmon overcome these problems, but these tools require a high quality, comprehensive reference transcripts dataset (RTD), which are rarely available in plants. RESULTS: A high-quality, non-redundant barley gene RTD and database (Barley Reference Transcripts - BaRTv1.0) has been generated. BaRTv1.0, was constructed from a range of tissues, cultivars and abiotic treatments and transcripts assembled and aligned to the barley cv. Morex reference genome (Mascher et al. Nature; 544: 427-433, 2017). Full-length cDNAs from the barley variety Haruna nijo (Matsumoto et al. Plant Physiol; 156: 20-28, 2011) determined transcript coverage, and high-resolution RT-PCR validated alternatively spliced (AS) transcripts of 86 genes in five different organs and tissue. These methods were used as benchmarks to select an optimal barley RTD. BaRTv1.0-Quantification of Alternatively Spliced Isoforms (QUASI) was also made to overcome inaccurate quantification due to variation in 5' and 3' UTR ends of transcripts. BaRTv1.0-QUASI was used for accurate transcript quantification of RNA-seq data of five barley organs/tissues. This analysis identified 20,972 significant differentially expressed genes, 2791 differentially alternatively spliced genes and 2768 transcripts with differential transcript usage. CONCLUSION: A high confidence barley reference transcript dataset consisting of 60,444 genes with 177,240 transcripts has been generated. Compared to current barley transcripts, BaRTv1.0 transcripts are generally longer, have less fragmentation and improved gene models that are well supported by splice junction reads. Precise transcript quantification using BaRTv1.0 allows routine analysis of gene expression and AS.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Hordeum/genética , Proteínas de Plantas/genética , Empalme Alternativo , Bases de Datos Genéticas , Regulación de la Expresión Génica de las Plantas , Análisis de Secuencia de ARN , Secuenciación del ExomaRESUMEN
Numerous genes that determine the outcome of plant-pathogen interactions are currently being discovered and include, for example, immune receptors, susceptibility factors and pathogen effectors and their host targets. Target enrichment sequencing provides a means to preferentially resequence these genes of interest without the need to first generate a genotype-specific genome assembly. The Basic Local Alignment Search Tool (BLAST), in combination with the here developed BLASTmap, can be used to design probes that specifically target such gene(s), either by using the target species or the closest related genus as a reference. BLAST is a ubiquitous tool in biological sequence analysis and a multitude of programs are available for the visualization of BLAST alignments. However, there are currently no dedicated programs for visual comparison of large-scale BLAST output attributes such as bit score. The need to quickly and efficiently compare many thousands of BLAST results led to the development of BLASTmap, an interactive web application created using the Shiny R package, customized for clustering and viewing BLAST results as an interactive heat map. Here we show an example of how BLASTmap was successfully applied to analyze custom DNA/RNA probe sequences and to visually determine that four probes are sufficient for the specific yet inclusive enrichment of the potato R2 disease resistance gene family.
Asunto(s)
Biología Computacional/métodos , Interacciones Huésped-Patógeno/genética , Programas Informáticos , Bases de Datos Genéticas , Interacciones Huésped-Patógeno/inmunología , Análisis de Secuencia de ADN , Interfaz Usuario-ComputadorRESUMEN
This chapter is designed to be a practical guide to using Tablet for the visualization of next/second-generation (NGS) sequencing data. NGS data is being produced more frequently and in greater data volumes every year. As such, it is increasingly important to have tools which enable biologists and bioinformaticians to understand and gain key insights into their data. Visualization can play a key role in the exploration of such data as well as aid in the visual validation of sequence assemblies and features such as single nucleotide polymorphisms (SNPs). We aim to show several use cases which demonstrate Tablet's ability to visually highlight various situations of interest which can arise in NGS data.
Asunto(s)
Mapeo Cromosómico/métodos , Biología Computacional/métodos , Genómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Navegador WebRESUMEN
BACKGROUND: Single Nucleotide Polymorphisms (SNPs) are widely used molecular markers, and their use has increased massively since the inception of Next Generation Sequencing (NGS) technologies, which allow detection of large numbers of SNPs at low cost. However, both NGS data and their analysis are error-prone, which can lead to the generation of false positive (FP) SNPs. We explored the relationship between FP SNPs and seven factors involved in mapping-based variant calling - quality of the reference sequence, read length, choice of mapper and variant caller, mapping stringency and filtering of SNPs by read mapping quality and read depth. This resulted in 576 possible factor level combinations. We used error- and variant-free simulated reads to ensure that every SNP found was indeed a false positive. RESULTS: The variation in the number of FP SNPs generated ranged from 0 to 36,621 for the 120 million base pairs (Mbp) genome. All of the experimental factors tested had statistically significant effects on the number of FP SNPs generated and there was a considerable amount of interaction between the different factors. Using a fragmented reference sequence led to a dramatic increase in the number of FP SNPs generated, as did relaxed read mapping and a lack of SNP filtering. The choice of reference assembler, mapper and variant caller also significantly affected the outcome. The effect of read length was more complex and suggests a possible interaction between mapping specificity and the potential for contributing more false positives as read length increases. CONCLUSIONS: The choice of tools and parameters involved in variant calling can have a dramatic effect on the number of FP SNPs produced, with particularly poor combinations of software and/or parameter settings yielding tens of thousands in this experiment. Between-factor interactions make simple recommendations difficult for a SNP discovery pipeline but the quality of the reference sequence is clearly of paramount importance. Our findings are also a stark reminder that it can be unwise to use the relaxed mismatch settings provided as defaults by some read mappers when reads are being mapped to a relatively unfinished reference sequence from e.g. a non-model organism in its early stages of genomic exploration.
Asunto(s)
Eucariontes/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Anotación de Secuencia Molecular/métodos , Polimorfismo de Nucleótido Simple/genética , Análisis de Secuencia de ADN/normas , Programas Informáticos , Arabidopsis/genética , Bases de Datos Genéticas , Genoma , Genómica/métodosRESUMEN
The low-recombining pericentromeric region of the barley genome contains roughly a quarter of the genes of the species, embedded in low-recombining DNA that is rich in repeats and repressive chromatin signatures. We have investigated the effects of pericentromeric region residency upon the expression, diversity and evolution of these genes. We observe no significant difference in average transcript level or developmental RNA specificity between the barley pericentromeric region and the rest of the genome. In contrast, all of the evolutionary parameters studied here show evidence of compromised gene evolution in this region. First, genes within the pericentromeric region of wild barley show reduced diversity and significantly weakened purifying selection compared with the rest of the genome. Second, gene duplicates (ohnolog pairs) derived from the cereal whole-genome duplication event ca. 60MYa have been completely eliminated from the barley pericentromeric region. Third, local gene duplication in the pericentromeric region is reduced by 29% relative to the rest of the genome. Thus, the pericentromeric region of barley is a permissive environment for gene expression but has restricted gene evolution in a sizeable fraction of barley's genes.
Asunto(s)
Evolución Molecular , Variación Genética , Genoma de Planta/genética , Hordeum/genética , Secuencia de Bases , Duplicación de Gen , Expresión Génica , Ontología de Genes , Heterocromatina/genética , Datos de Secuencia Molecular , Recombinación Genética , Análisis de Secuencia de ARNRESUMEN
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.
Asunto(s)
Gráficos por Computador , Presentación de Datos , Bases de Datos Genéticas/estadística & datos numéricos , Animales , Biología Computacional , Genómica/estadística & datos numéricos , Secuenciación de Nucleótidos de Alto Rendimiento/estadística & datos numéricos , Humanos , Internet , Análisis de Secuencia/estadística & datos numéricos , Programas InformáticosRESUMEN
UNLABELLED: 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/.
Asunto(s)
Biología Computacional/métodos , Gráficos por Computador , Genómica/métodos , Programas Informáticos , Genoma , Mapeo Físico de Cromosoma , Interfaz Usuario-ComputadorRESUMEN
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 .
Asunto(s)
Gráficos por Computador , Genotipo , Programas Informáticos , Mapeo Cromosómico , Polimorfismo de Nucleótido Simple , Sitios de Carácter CuantitativoRESUMEN
SUMMARY: Tablet is a lightweight, high-performance graphical viewer for next-generation sequence assemblies and alignments. Supporting a range of input assembly formats, Tablet provides high-quality visualizations showing data in packed or stacked views, allowing instant access and navigation to any region of interest, and whole contig overviews and data summaries. Tablet is both multi-core aware and memory efficient, allowing it to handle assemblies containing millions of reads, even on a 32-bit desktop machine. AVAILABILITY: Tablet is freely available for Microsoft Windows, Apple Mac OS X, Linux and Solaris. Fully bundled installers can be downloaded from http://bioinf.scri.ac.uk/tablet in 32- and 64-bit versions.