10 results on '"Paulo Rapazote-Flores"'
Search Results
2. BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq
- Author
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Paulo Rapazote-Flores, Micha Bayer, Linda Milne, Claus-Dieter Mayer, John Fuller, Wenbin Guo, Pete E. Hedley, Jenny Morris, Claire Halpin, Jason Kam, Sarah M. McKim, Monika Zwirek, M. Cristina Casao, Abdellah Barakate, Miriam Schreiber, Gordon Stephen, Runxuan Zhang, John W. S. Brown, Robbie Waugh, and Craig G. Simpson
- Subjects
Barley ,Reference transcript dataset ,Transcriptome ,Differential gene expression ,Differential alternative splicing ,Biotechnology ,TP248.13-248.65 ,Genetics ,QH426-470 - Abstract
Abstract 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.
- Published
- 2019
- Full Text
- View/download PDF
3. Barley SIX-ROWED SPIKE3 encodes a putative Jumonji C-type H3K9me2/me3 demethylase that represses lateral spikelet fertility
- Author
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Hazel Bull, M. Cristina Casao, Monika Zwirek, Andrew J. Flavell, William T. B. Thomas, Wenbin Guo, Runxuan Zhang, Paulo Rapazote-Flores, Stylianos Kyriakidis, Joanne Russell, Arnis Druka, Sarah M. McKim, and Robbie Waugh
- Subjects
Science - Abstract
The VRS genes of barley control the fertility of the lateral spikelets on the barley inflorescence. Here, Bull et al. show that VRS3 encodes a putative Jumonji C-type histone demethylase that regulates expression of other VRS genes, and genes involved in stress, hormone and sugar metabolism.
- Published
- 2017
- Full Text
- View/download PDF
4. Development and Evaluation of a Barley 50k iSelect SNP Array
- Author
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Micha M. Bayer, Paulo Rapazote-Flores, Martin Ganal, Pete E. Hedley, Malcolm Macaulay, Jörg Plieske, Luke Ramsay, Joanne Russell, Paul D. Shaw, William Thomas, and Robbie Waugh
- Subjects
barley ,SNP ,genotyping chip ,iSelect ,exome capture ,Plant culture ,SB1-1110 - Abstract
High-throughput genotyping arrays continue to be an attractive, cost-effective alternative to sequencing based approaches. We have developed a new 50k Illumina Infinium iSelect genotyping array for barley, a cereal crop species of major international importance. The majority of SNPs on the array have been extracted from variants called in exome capture data of a wide range of European barley germplasm. We used the recently published barley pseudomolecule assembly to map the exome capture data, which allowed us to generate markers with accurate physical positions and detailed gene annotation. Markers from an existing and widely used barley 9k Infinium iSelect array were carried over onto the 50k chip for backward compatibility. The array design featured 49,267 SNP markers that converted into 44,040 working assays, of which 43,461 were scorable in GenomeStudio. Of the working assays, 6,251 are from the 9k iSelect platform. We validated the SNPs by comparing the genotype calls from the new array to legacy datasets. Rates of agreement averaged 98.1 and 93.9% respectively for the legacy 9k iSelect SNP set (Comadran et al., 2012) and the exome capture SNPs. To test the utility of the 50k chip for genetic mapping, we genotyped a segregating population derived from a Golden Promise × Morex cross (Liu et al., 2014) and mapped over 14,000 SNPs to genetic positions which showed a near exact correspondence to their known physical positions. Manual adjustment of the cluster files used by the interpreting software for genotype scoring improved results substantially, but migration of cluster files between sites led to a deterioration of results, suggesting that local adjustment of cluster files is required on a site-per-site basis. Information relating to the markers on the chip is available online at https://ics.hutton.ac.uk/50k.
- Published
- 2017
- Full Text
- View/download PDF
5. BaRTv1.0: an improved barley reference transcript dataset to determine accurate changes in the barley transcriptome using RNA-seq
- Author
-
Micha Bayer, Sarah M. McKim, Wenbin Guo, John L. Fuller, Craig G. Simpson, Paulo Rapazote-Flores, Claus-Dieter Mayer, Pete E. Hedley, Miriam Schreiber, Jason Kam, Monika Zwirek, M. Cristina Casao, Claire Halpin, Jenny Morris, Gordon Stephen, Abdellah Barakate, Robbie Waugh, Runxuan Zhang, John W. S. Brown, and Linda Milne
- Subjects
Gene isoform ,0106 biological sciences ,Untranslated region ,Differential alternative splicing ,lcsh:QH426-470 ,lcsh:Biotechnology ,RNA-Seq ,Computational biology ,Biology ,01 natural sciences ,Transcriptome ,03 medical and health sciences ,Gene Expression Regulation, Plant ,Barley ,lcsh:TP248.13-248.65 ,Databases, Genetic ,Exome Sequencing ,Gene expression ,Splice junction ,Genetics ,Gene ,Differential gene expression ,Plant Proteins ,030304 developmental biology ,2. Zero hunger ,0303 health sciences ,Sequence Analysis, RNA ,Gene Expression Profiling ,Spliced Genes ,food and beverages ,Hordeum ,Alternative Splicing ,lcsh:Genetics ,RNA splicing ,Reference transcript dataset ,DNA microarray ,Research Article ,010606 plant biology & botany ,Biotechnology ,Reference genome - Abstract
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.
- Published
- 2019
6. EORNA, a barley gene and transcript abundance database
- Author
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Robbie Waugh, Linda Milne, Paulo Rapazote-Flores, Micha Bayer, Craig G. Simpson, and Claus-Dieter Mayer
- Subjects
0106 biological sciences ,Statistics and Probability ,Data Descriptor ,Transcription, Genetic ,Science ,RNA-Seq ,Biology ,Library and Information Sciences ,computer.software_genre ,Genes, Plant ,01 natural sciences ,Education ,03 medical and health sciences ,Abundance (ecology) ,Gene Expression Regulation, Plant ,Reference Values ,Gene expression ,Databases, Genetic ,Transcriptomics ,Gene ,030304 developmental biology ,2. Zero hunger ,Regulation of gene expression ,0303 health sciences ,Database ,Models, Genetic ,fungi ,Alternative splicing ,food and beverages ,Hordeum ,Computer Science Applications ,Gene nomenclature ,Alternative Splicing ,13. Climate action ,Transcription (software) ,Statistics, Probability and Uncertainty ,Genetic databases ,computer ,010606 plant biology & botany ,Information Systems - Abstract
A high-quality, barley gene reference transcript dataset (BaRTv1.0), was used to quantify gene and transcript abundances from 22 RNA-seq experiments, covering 843 separate samples. Using the abundance data we developed a Barley Expression Database (EORNA*) to underpin a visualisation tool that displays comparative gene and transcript abundance data on demand as transcripts per million (TPM) across all samples and all the genes. EORNA provides gene and transcript models for all of the transcripts contained in BaRTV1.0, and these can be conveniently identified through either BaRT or HORVU gene names, or by direct BLAST of query sequences. Browsing the quantification data reveals cultivar, tissue and condition specific gene expression and shows changes in the proportions of individual transcripts that have arisen via alternative splicing. TPM values can be easily extracted to allow users to determine the statistical significance of observed transcript abundance variation among samples or perform meta analyses on multiple RNA-seq experiments. * Eòrna is the Scottish Gaelic word for Barley., Measurement(s) gene expression Technology Type(s) transcription profiling assay Factor Type(s) Genotype • Abiotic Stress • Developmental stage • Tissue • Biotic stress Sample Characteristic - Organism Hordeum vulgare Sample Characteristic - Environment sodium chloride salt • drought • increased temperature • decreased temperature Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.13643387
- Published
- 2020
7. EoRNA, a barley gene and transcript abundance database
- Author
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Paulo Rapazote-Flores, Linda Milne, Claus-Dieter Mayer, Robbie Waugh, Micha Bayer, and Craig G. Simpson
- Subjects
Gene nomenclature ,Database ,Abundance (ecology) ,On demand ,Alternative splicing ,Gene expression ,food and beverages ,RNA ,Biology ,computer.software_genre ,computer ,Gene - Abstract
A high-quality, barley gene reference transcript dataset (BaRTv1.0), was used to quantify gene and transcript abundances from 22 RNA-seq experiments, covering 843 separate samples. Using the abundance data we developed a Barley Expression Database (EoRNA* – Expression of RNA) to underpin a visualisation tool that displays comparative gene and transcript abundance data on demand as transcripts per million (TPM) across all samples and all the genes. EoRNA provides gene and transcript models for all of the transcripts contained in BaRTV1.0, and these can be conveniently identified through either BaRT or HORVU gene names, or by direct BLAST of query sequences. Browsing the quantification data reveals cultivar, tissue and condition specific gene expression and shows changes in the proportions of individual transcripts that have arisen via alternative splicing. TPM values can be easily extracted to allow users to determine the statistical significance of observed transcript abundance variation among samples or perform meta analyses on multiple RNA-seq experiments. * Eòrna is the Scottish Gaelic word for Barley
- Published
- 2020
8. High-Resolution RT-PCR Analysis of Alternative Barley Transcripts
- Author
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Craig G, Simpson, John, Fuller, Paulo, Rapazote-Flores, Claus-Dieter, Mayer, Cristiane P G, Calixto, Linda, Milne, Pete E, Hedley, Clare, Booth, Robbie, Waugh, and John W S, Brown
- Subjects
Alternative Splicing ,Analysis of Variance ,DNA, Complementary ,Organ Specificity ,RNA, Plant ,Reverse Transcriptase Polymerase Chain Reaction ,RNA ,Hordeum ,Genes, Plant - Abstract
Assembly of the barley genome and extensive use of RNA-seq has resulted in an abundance of gene expression data and the recognition of wide-scale production of alternatively spliced transcripts. Here, we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) that confirms the accuracy of alternatively spliced transcripts from RNA-seq and allows quantification of changes in the proportion of splice isoforms between different experimental conditions, time points, tissues, genotypes, ecotypes, and treatments. By validating a selection of barley genes, use of the panel gives confidence or otherwise to the genome-wide global changes in alternatively spliced transcripts reported by RNA-seq. This simple assay can readily be applied to perform detailed transcript isoform analysis for any gene in any species.
- Published
- 2018
9. High-Resolution RT-PCR Analysis of Alternative Barley Transcripts
- Author
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Clare Booth, Claus-Dieter Mayer, Pete E. Hedley, Paulo Rapazote-Flores, Cristiane P. G. Calixto, John L. Fuller, John W. S. Brown, Robbie Waugh, Linda Milne, and Craig G. Simpson
- Subjects
0106 biological sciences ,0301 basic medicine ,Gene isoform ,Alternative splicing ,RNA-Seq ,Computational biology ,Biology ,01 natural sciences ,Genome ,03 medical and health sciences ,030104 developmental biology ,Rt pcr analysis ,Gene expression ,Genotype ,Gene ,010606 plant biology & botany - Abstract
Assembly of the barley genome and extensive use of RNA-seq has resulted in an abundance of gene expression data and the recognition of wide-scale production of alternatively spliced transcripts. Here, we describe in detail a high-resolution reverse transcription-PCR based panel (HR RT-PCR) that confirms the accuracy of alternatively spliced transcripts from RNA-seq and allows quantification of changes in the proportion of splice isoforms between different experimental conditions, time points, tissues, genotypes, ecotypes, and treatments. By validating a selection of barley genes, use of the panel gives confidence or otherwise to the genome-wide global changes in alternatively spliced transcripts reported by RNA-seq. This simple assay can readily be applied to perform detailed transcript isoform analysis for any gene in any species.
- Published
- 2018
10. The Expressed Portion of the Barley Genome
- Author
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Roberto A. Barrero, Pete E. Hedley, Robbie Waugh, Micha Bayer, Matthew I. Bellgard, Runxuan Zhang, Andrew J. Flavell, Paulo Rapazote-Flores, and Craig G. Simpson
- Subjects
Mechanism (biology) ,microRNA ,Alternative splicing ,Gene expression ,Gene regulatory network ,Computational biology ,Biology ,Gene ,Psychological repression ,Genome - Abstract
In this chapter, we refer to the expressed portion of the barley genome as the relatively small fraction of the total cellular DNA that either contains the genes that ultimately produce proteins, or that directly/indirectly controls the level, location and/or timing of when these genes are expressed and proteins are produced. We start by describing the dynamics of tissue and time-dependent gene expression and how common patterns across multiple samples can provide clues about gene networks involved in common biological processes. We then describe some of the complexities of how a single mRNA template can be differentially processed by alternative splicing to generate multiple different proteins or provide a mechanism to regulate the amount of functional gene product in a cell at a given point in time. We extend our analysis, using a number of biological examples, to address how diverse families of small non-coding microRNAs specifically regulate gene expression, and complete our appraisal by looking at the physical/molecular environment around genes that can result in either the promotion or repression of gene expression. We conclude by assessing some of the issues that remain around our ability to fully exploit the depth and power of current approaches for analysing gene expression and propose improvements that could be made using new but available sequencing and bioinformatics technologies.
- Published
- 2018
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