ORDER

Oilseed Rape Developmental Expression Resource


Brassica napus


The Oilseed Rape Developmental Expression Resource (ORDER) was created to provide access to the developmental transcriptome of Brassica napus during the floral transition.

Guide

The About tab presents information about how the experiment was carried out, such as which Brassica napus varieties were grown, how the samples were collected and how gene expression levels were quantified.

The How To Use tab explains how to use the website to search the database. This is the place to go if you have questions about the user interface of the website.

The final three tabs (Search, BLAST Search and Table) are the tabs you'll be using to search the database.

Contact

Please could you direct all enquiries, bug reports and interesting findings which relate to this dataset to Marc Jones.

AionPlot

ORDER is an instance of the time series visualization and dissemination platform AionPlot.

About

This is a web app to facilitate interrogation of time series data collected from Brassica napus. The data was collected from the cultivars Tapidor (a semi-winter cultivar) and Westar (a spring cultivar) with the aim of deciphering how the flowering time gene regulatory network is constructed, and how it differs from the network elucidated in Arabidopsis thaliana. An overview of the plant growth regime and data processing pipeline is provided below, but for further details please consult our published work on the project (Jones et al. 2018).

Growth Details

Plants were germinated and grown in a glasshouse for two weeks before being transferred to a controlled environment room to undergo vernalization. Plants from both the semi-winter and spring varieties were vernalized to allow more accurate between variety comparisons to be made. After six weeks of vernalization, plants were removed from the controlled environment rooms and grown in a glasshouse until the plants began to flower.

Sampling Details

At each time point, trays of plants were sampled and the biological tissue pooled. The first true leaf and the apex of each plant was taken and frozen initially on dry ice and then in liquid nitrogen. While on dry ice, apicies were dissected to remove as much leaf and stem tissue as possible while leaving the apex intact.

RNA Extraction Details

Tissue was ground and RNA extracted using the OMEGA bio-tek E.Z.N.A.® Plant RNA Kit in accordance with the manufacturer instructions. An on-column DNase step was performed using a compatible product and method provided by OMEGA bio-tek. RNA quality assurance and sequencing was conducted by The Earlham Institute, Norwich.

Expression Quantification

The raw expression reads were analysed using the Tuxedo Suite of tools. These consist of Bowtie, TopHat and Cufflinks. Bowtie is an alignment algorithm which TopHat uses to align reads to a reference sequence. TopHat is a splice-aware aligner, which is able to align sequencing reads that span a splice site to genomic reference sequence. Finally, Cufflinks takes the positions of the aligned sequencing reads and performs quantification to give an expression level for each identified transcript.

The data available in this web app was generated using the available Brassica napus genome sequence as a reference sequence. This sequence was generated from sequencing the Brassica napus cultivar Darmor-bzh (Chalhoub et al. 2014). Gene models were determined directly from the sequencing data using a combination of Augustus and Cufflinks.

Cufflinks uses a statistical model to estimate the uncertainty of each expression measurement. This model takes into account biological and technical variation through the use of repeat experiments, as well as read mapping uncertainty. Read mapping uncertainty arises due to sequencing reads having multiple potential positions within the reference genome from which they could originate. Repeat experiments, using different tissue pools sampled simultaneously and separate sequencing runs, were conducted for the majority of time points. Cufflinks uses these repeat samples to estimate the error for samples without replication. The complete sampling scheme used is available in Jones et al. (2018).

Mappings to published gene models

The Brassica napus gene models used to generate the dataset presented in ORDER used the collected RNA-Seq reads to inform gene model prediction. To allow these novel gene models to be useful to the community, ORDER contains mappings from published gene models to the gene models used in ORDER. The published gene models include:

  • Arabidopsis thaliana - TAIR10 gene models
  • Brassica oleracea - BOL gene models from release 43 of Ensembl Plants
  • Brassica rapa - BRAD version 3.0
  • Brassica napus - Pantranscriptome presented in He et al. (2015). To prevent confusion with duplicate gene model names, the gene models from the Pantranscriptome are prefixed with "Pan_".

How To Use

In order to search the database, please use the Search, BLAST Search and Table tabs.

Search

The Search tab allows users to plot the expression of Brassica napus genes across developmental time. To search for Brassica napus genes, type the name or abbreviation of a gene of interest and select the gene from the dropdown list. Although you may search for Brassica napus genes directly, mappings to a number of published gene model sets are also available and can be searched for. For a list of the included gene model mappings, please visit the About tab. You may enter multiple genes at once. Brassica napus genes which show homology to your genes of interest will be displayed below the search box. To plot the expression of a Brassica napus gene, click on its name. Hovering over the button or the name of the gene in the plot legend will reveal the Brassica napus chromosome where that gene is located. The colour of the button indicates the homology status of the Brassica napus gene. If the button is green
that indicates that that Brassica napus gene has greatest homology to the selected gene model. If the button is yellow
that indicates that that Brassica napus gene has greatest homology to a splice variant of the selected gene model. Unless you have searched for a Brassica napus gene directy, white buttons
indicate that that Brassica napus gene has greatest homology to another gene model. These button colours are intended as a guide to help aid users, but should be scrutinised to ensure accuracy.

BLAST Search

ORDER may also be queried using a DNA sequence. To do this, copy and paste your sequence of interest into the text box on the BLAST Search tab. The information box on that page will indicate the number of Brassica napus genes that show homology to your sequence. To plot the expression of these genes, navigate back to the Search tab. The genes that show homology to the sequence you entered in the BLAST Search tab will be displayed as 'BLAST Hits'.

Table

Displays a table of details about the Brassica napus genes currently selected in the Search tab. Clicking the symbol will expand the row to reveal further homology information about that gene. The homology information is a table of all gene model mappings for that particular Brassica napus gene. The row colouring matches the button colouring detailed above in the section explaining the Search tab.

BLAST Search

Table