RNA-Seq analysis with AskOmics Interactive Tool
OverviewQuestions:Objectives:
How to integrate RNA-Seq results with other datasets using AskOmics?
How to query these datasets to answer a biological question?
How to exploit a distant SPARQL endpoint to retrieve information?
Requirements:
Launch an AskOmics Interactive Tool
Integrate RNA-Seq and reference datasets into AskOmics
Create a complex query to get over-expressed genes, their location on the reference genome, and check if they are included in a known QTL
Complete the query to get the human homologs and their location using neXtProt database
- Introduction to Galaxy Analyses
- Sequence analysis
- Quality Control: slides slides - tutorial hands-on
- Mapping: slides slides - tutorial hands-on
- Transcriptomics
- 2: RNA-seq counts to genes: tutorial hands-on
Time estimation: 2 hoursSupporting Materials:Last modification: Oct 18, 2022
Introduction
AskOmics is a web application for data integration and querying using the Semantic Web technologies. It helps users to convert multiple data sources (CSV/TSV files, GFF and BED annotation) into “RDF triples” and store them in a specific kind of database: an “RDF triplestore”. Under this form, data can then be queried using a specific language: “SPARQL”. AskOmics hides the complexity of these technologies and allows to perform complex queries using a user-friendly interface.
AskOmics comes in useful for cross-referencing results datasets with various reference data. For example, in RNA-Seq studies, we often need to filter the results on the fold change and the p-value, to get the most significant differentially expressed genes. If you are studying a particular phenotype and already know the position of some QTL associated to this phenotype, you would then want to find the positions of the differentially expressed genes and determine which gene is located within one of those QTL. Finally, you would want to know if these genes have human homologs, and use the neXtProt database to get the location of the proteins coded by the homologs. The whole process involves several tools to parse and manipulate the different data format, and to map datasets on each other. AskOmics offer a solution to 1) automatically convert the multiple formats to RDF, 2) use a user-friendly interface to perform complex SPARQL queries on the RDF datasets to find the genes you are interested in, and 3) connect external SPARQL databases and link external data with your own.
In this tutorial, we will use results from a differential expression analysis. This file is provided for you below. You could also generate the file yourself, by following the RNA-Seq counts to gene tutorial. The file used here was generated from limma-voom but you could use a file from any RNA-seq differential expression tool, such as edgeR or DESeq2, as long as it contains the required columns (see below).
The differentially expressed results will be linked to the official mouse genome annotation, in general feature format (GFF). The file provided is a subset of the mouse annotation (GRCm38.p6) obtained from Ensembl.
We will use a file containing quantitative trait loci (QTL) information, to find if our differentially expressed genes are located inside a known QTL. This file is a subset of a query performed on Mouse Genome Informatics.
A file containing all homologies between mouse and human will be used to get the human homolog genes. This file is provided by MGI.
In the differentially expressed file, and the homologs file, gene are described by a symbol (e.g. Pwgrq10). However, in the annotation file and neXtProt database, gene are represented by Ensembl id (e.g. ENSMUSG00000025969). To link the 2 datasets, we will need a file to map the gene symbol with Ensembl id. This file provided for you was previously generated with an AskOmics query on the mouse annotation file and the homolog file.
To link the human gene with neXtProt database, we will use the RDF abstraction of neXtProt. This file was obtained using the Abstractor tool.
During the integration step, AskOmics builds an RDF description of the data: the abstraction. This abstraction is used to explore the data and build the query. AskOmics can also integrate abstraction of distant endpoint. Abstraction are obtained using abstractor, a python package to generate RDF abstractions from distant endpoints. The query builder interface is used to create a path through the abstraction of each ressources. The path is converted to a SPARQL query that is sent to the multiple SPARQL endpoint.
AgendaIn this tutorial, we will cover:
Preparing the inputs
We will use four files for this analysis:
- Differentially expressed results file (genes in rows, and 4 required columns: identifier (ENTREZID), gene symbol (SYMBOL), log fold change (logFC) and adjusted P values (adj.P.Val))
- Reference genome annotation file (in GFF format)
- QTL file (QTL in rows, with 5 required columns: identifier, chromosome, start, end and name)
- Homolog file (TSV of 13 columns including homolog id, organism name and gene symbol)
- Correspondence file between gene symbol and Ensembl id (TSV of 3 columns: symbol, the corresponding Ensembl id (mouse and human)
- neXtProt abstraction (RDF data description of neXtProt database in turtle format)
Import data
Hands-on: Data upload
Create a new history for this RNA-seq exercise e.g.
RNA-seq AskOmics
Click the new-history icon at the top of the history panel.
If the new-history is missing:
- Click on the galaxy-gear icon (History options) on the top of the history panel
- Select the option Create New from the menu
- Click on Unnamed history (or the current name of the history) (Click to rename history) at the top of your history panel
- Type the new name
- Press Enter
Import the files.
To import the files, there are two options:
- Option 1: From a shared data library if available (ask your instructor)
- Option 2: From Zenodo
- Copy the link location
Open the Galaxy Upload Manager (galaxy-upload on the top-right of the tool panel)
- Select Paste/Fetch Data
Paste the link into the text field
Press Start
- Close the window
As an alternative to uploading the data from a URL or your computer, the files may also have been made available from a shared data library:
- Go into Shared data (top panel) then Data libraries
- Navigate to the correct folder as indicated by your instructor
- Select the desired files
- Click on the To History button near the top and select as Datasets from the dropdown menu
- In the pop-up window, select the history you want to import the files to (or create a new one)
- Click on Import
You can paste the links below into the Paste/Fetch box:
https://zenodo.org/record/2529117/files/limma-voom_luminalpregnant-luminallactate https://zenodo.org/record/3950862/files/Mus_musculus.GRCm38.98.subset.gff3 https://zenodo.org/record/3950862/files/Symbol.tsv https://zenodo.org/record/3950862/files/MGIBatchReport_Qtl_Subset.txt https://zenodo.org/record/3950862/files/HOM_MouseHumanSequence.rpt https://zenodo.org/record/3950862/files/nextprot_abstraction.ttl
- Rename the files using the galaxy-pencil (pencil) icon.
- limma-voom_luminalpregnant-luminallactate to
DE results
- Mus_musculus.GRCm38.98.subset.gff3 to
Mus musculus annotation
- Symbol.tsv to
Gene Symbols
- MGIBatchReport_Qtl_Subset.txt to
QTL
- HOM_MouseHumanSequence.rpt to
Homolog groups
- nextprot_asbtraction.ttl to
neXtProt abstraction
- Check every datatype.
- DE results:
tabular
- Mus musculus annotation:
gff
- Gene Symbol:
tabular
- QTL:
tabular
- Homolog groups:
tabular
- neXtprot abstraction:
ttl
If the datatypes are wrong, please change it.
- Click on the galaxy-pencil pencil icon for the dataset to edit its attributes
- In the central panel, click on the galaxy-chart-select-data Datatypes tab on the top
- Select your desired datatype
- tip: you can start typing the datatype into the field to filter the dropdown menu
- Click the Save button
Click on the galaxy-eye (eye) icon and take a look at the uploaded files.
Two step are necessary to get our data converted into RDF triples. The first step is to upload the Galaxy datasets into the AskOmics server. The second step is to integrate the uploaded data into the RDF triplestore.
Upload inputs into AskOmics
We will first launch an AskOmics interactive tool, and upload the data into it.
Launch AskOmics Interactive Tool
Hands-on: Launch AskOmics IT
- AskOmics a visual SPARQL query builder tool to launch the Interactive Tool
- param-file “Datasets to load into AskOmics”:
DE results
,Mus musculus annotation
,Gene Symbols
,QTL
,Homolog groups
andneXtProt abstraction
Wait a few seconds (or minutes if computing resources are busy) for AskOmics to be ready to use. A view link should appear in the confirmation box just after clicking on the Execute button.
AskOmics is an Interactive tool. It means that when you launch it, it will stay in running state (yellow background) in your History. As long as it stays in this running state, you can access it by looking in the “User” > “Active Interactive Tools” menu (click on its name to view it). When you no longer need it, you can stop it by deleting it from your history, or using the “Stop” button in the “User” > “Active Interactive Tools” page.
Keep in mind that as long as this tool runs, it uses computing resources, so don’t forget to stop it when you no longer have use for it.
Once the AskOmics Interactive Tool is ready, you should see a start page looking like this:
You can see that there is no data available yet. It’s because data needs to be integrated: it is the next step.
Integrate input files into AskOmics
AskOmics conversion into RDF is called integration.
On the Files page (link at the top of the page), you will see the files you uploaded from Galaxy. We will now integrate all these files.
Hands-on: Integrate data
- Got to the Files page
- Select all the input files
- Click on the Integrate button
You will land on the Integrate page that shows a preview of the data present in each selected file, depending of its data type.
Integrate GFF files
The GFF preview shows the entities that the file contains. We need to select the entities we want to be integrated.
The Mus musculus annotation
file we’re using contains gene and mRNA entities, and we will need both in the rest of the tutorial.
Hands-on: Integrate `Mus musculus annotation`
- Search for
Mus musculus annotation (preview)
- Select
gene
andmRNA
- Click on the Integrate (private dataset) button
Integration of tabular (TSV) files
The TSV preview shows an HTML table representing the first lines of the TSV file. During integration, AskOmics will convert the file using the header.
The first column of a TSV file will be the entity name. Other columns of the file will be attributes of the entity. Labels of the entity and attributes will be set by the header. Each label can be edited by clicking on it.
Entity and attributes can have special types. The types are defined with the select box below the header. An entity can be a start entity or an entity. A start entity means that the entity may be used to start a query on the AskOmics homepage.
Attributes can take the following types:
- Numeric: if all the values of the column are numeric
- Text: if all the values are strings
- Category: if there is a limited number of repeated values (e.g. ‘green’, ‘yellow’ and ‘red’, each one found in multiple lines)
If the entity describes a locatable element on a genome:
- Reference: chromosome
- Strand: strand
- Start: start position
- End: end position
A column can also represent a relation between the entity to another. In this case, the header have to be named relationName@TargetedEntity
and the type Directed or Symetric relation. A Directed relation is a relation from this entity to the targeted one (e.g. A is B’s father, but B is not A’s father). A Symetric relation is a relation that works in both directions (e.g. A loves B, and B loves A).
Hands-on: Integrate `DE results`
- Search for
DE results (preview)
- Edit attribute names and types:
- change
ENTREZ ID
toDifferential Expression
and set type to start entity- change
SYMBOL
tolinkedTo@Gene Symbol
and set type to Symetric relation- change
GENENAME
toname
and set type to text- Keep the other column names and set their types to numeric
- Integrate (private dataset)
Hands-on: Integrate `Gene symbols`
- Search for
Gene symbols (preview)
- Edit attribute names and types:
- change
symbol
toGene Symbol
and set type to entity- Set
to mouse genes@gene
type to Symetric relation- Click on the Integrate (private dataset) button
Hands-on: Integrate `QTL`
- Search for
QTL (preview)
- Edit attribute names and types:
- change
Input
toQTL
and set type to start entity- set
Chr
type to Reference- set
Start
type to Start- set
End
type to End- Click on the Integrate (private dataset) button
Hands-on: Integrate `Homolog groups`
- Search for
Homolog groups (preview)
- Edit attribute names and types:
- change
HomoloGene ID
toHomolog Group
and set type to start entity- set
Common Organism Name
type to category- change
Symbol
tolinkedTo@Gene Symbol
and set type to Directed relation- Keep the other column names and set their types to text
- Click on the Integrate (private dataset) button
Integration of RDF files
The last dataset we want to integrate is the neXtProt abstraction. This file contains some RDF data that instructs AskOmics how to communicate with a remote RDF database containing neXtProt data.
Hands-on: Integrate `neXtProt abstraction`
- Search for
neXtProt abstraction (preview)
- Check that Distant endpoint is set to
https://sparql.nextprot.org/sparql
in advanced options- Click on the Integrate (private dataset) button
Monitor integration
Integration can take some time depending on the file size. The Datasets page shows the progress.
Hands-on: track integration progress
- Go to the Dataset page
- Wait for all datasets to be in success state
Query
Once all the data of interest is integrated (converted to RDF), its time to query them. Querying RDF data is done by using the SPARQL language. Fortunately, AskOmics provides a user-friendly interface to build SPARQL queries without having to learn the SPARQL language.
Query builder overview
Simple query
The first step to build a query is to choose a start point for the query.
Hands-on: Start a query
- Go to the Ask! page
- Select the Differential Expression entity
- Start!
Once the start entity is chosen, the query builder is displayed.
The query builder is composed of a graph. Nodes represents entities and links represents relations between entities. The selected entity is surrounded by a red circle. Links and other entities are dotted and lighter because there are not instantiated.
On the right, attributes of the selected entity are displayed as attribute boxes. Each box has an eye icon: an opened eye means the attribute will be displayed on the results.
Hands-on: Ask for all Differential Expression and display some attributes
- Display
logFC
andadj.P.val
by clicking on the eye icon- Run & preview
Run & preview launch the query with a limit of 30 rows returned. We use this button to get an idea of the results returned.
Filter on attributes
Next query will search for all over-expressed genes. Genes are considered over-expressed if the log fold change is > 2. We are only interested by significant results (Adj P value ≤ 0.05)
Back to the query builder,
Hands-on: Filter attributes to get significant over-expressed genes
- Filter
logFC
with> 2
- Filter
adj.P.val
with≤ 0.05
- Run & preview
The preview shows only significantly over-expressed genes.
Filter on relations
Now that we have found our genes of interest, we will link these genes to the reference genome to get information about their location.
To constraint on relation, we have to click on suggested nodes, linked to our entity of interest.
Hands-on: Link `De results` to `gene` using `Gene Symbol`
- First, hide
Label
,logFC
andadj.P.val
ofDifferential Expression
using the eye icon- Instantiate
Gene Symbol
by clicking on the suggested node, and hide hisLabel
using the eye icon- Instantiate
gene
by clicking on thegene
node- Run & preview
Results now show the Ensembl id of our over-expressed genes. We have now access to all the information about the gene
entity contained in the GFF file. For example, we can filter on chromosome and display chromosome and strand to get information about gene location.
Hands-on: Filter `gene`
- Show
reference
andstrand
using the eye icon- Filter
reference
by selectingX
chromosome- Filter
strand
by selecting+
strand- Run & preview
Query on the position of elements on the genome.
AskOmics is able to perform special queries between entities that are locatable. These queries are:
- Entities overlapping another one
- Entities included in another entity
The FALDO ontology describes sequence feature positions and regions. AskOmics uses FALDO ontology to represent entity positions. GFF are using FALDO, as well as TSV entities with chromosome, strand, start and end.
On the query builder interface, locatable entities are represented with a green circle and relations based on location are represented as green arrow.
Hands-on: Filter `gene`
- First, remove the reference filter (unselect
X
usingctrl
+click
)- Remove the strand filter (unselect
+
usingctrl
+click
)- Hide
reference
strand
using the eye- Instantiate
QTL
- Click on the link between
gene
andQTL
to edit the relation- Check that the relation is
gene
included in
QTL
on the same reference
withstrict
ticked- Run & preview
To go further, we can filter on QTL
to refine the results.
Hands-on: Filter `gene`
- Go back to the
QTL
node- Show the
Name
attribute using the eye icon- Filter the name with a
regexp
withgrowth
- Run & preview
From now, our query is “All Genes that are over-expressed (logFC > 2 and FDR ≤ 0.05) and located on a QTL that is related to growth”. We can save this results with the Run & save button.
Hands-on: Save a result
- Run & save
Use neXtProt distant data to refine results
neXtProt is a comprehensive human-centric discovery platform, offering its users a seamless integration of and navigation through protein-related data. It offer a SPARQL endpoint that can be interrogated with AskOmics.
Since we added the neXtProt abstraction into our AskOmics instance, we can link our data to neXtProt.
Hands-on: Find human homolog genes
- Go back to the
gene
node- instantiate
Gene Symbol
and hide hisLabel
- Instantiate
Homolog Group
, hide his label and filter hisCommon Organism Name
withhuman
- From
Homolog Group
, instantiate anotherGene Symbol
and hide hisLabel
- Finally, follow the
to neXtProt Gene
link and instantiateGene
(with a capital G)- Run & preview
The query we’ve just built asks for the human homologs of our over-expressed genes. We use the Gene Symbol
to get information from the neXtProt database. AskOmics converts the query into small SPARQL subqueries and send them to the local database and to the remote neXtProt endpoint.
Now we are linked to the neXtProt database, we can obtain information about the proteins encoded by these genes, as well as their location in the cell.
Hands-on: Get the protein and their location
- Instantiate
Entry
- Instantiate
Isoform
and hide theLabel
- Many nodes are connected to
Isoform
. Use the Filter links field to filter nodes linked with a link namedlocation
- Instantiate the
Subcellular Location
node and hideUri
- Use the Filter node field to filter nodes with “location” in their name
- Instantiate
Uniprot subcellular Location CV
(you can use the node filter to clear up the screen)- Run & preview
Finally, our query is “All genes that are over-expressed and located on a QTL that is related to growth, their human homologs and the location of the proteins coded by this genes”. We will save it to the results.
Hands-on: Save a result
- Run & save
Results management
The results page displays the saved queries. Queries are sorted by creation date. At the end of the table, action buttons can be used to preview the result, download or send it to Galaxy history.
Hands-on: Edit query name
- Go to the Results page
- Use the Preview button to check the result
- Click on the name to rename the two query with
Over-expressed genes on a growth QTL
andOver-expressed genes on a growth QTL, their human homologs and protein location
(press enter key to validate)
The Action column contain button to perform certain action:
- Preview: show a results preview on the bottom of the table
- Download: Download the results (TSV file)
- Edit: Edit the query with the query builder
- SPARQL: edit the query with a SPARQL editor for advanced users
- Send results to Galaxy: send the results (TSV file) to the most recently used Galaxy history
- Send query to Galaxy: send the query representation (json file) to the most recently used Galaxy history
Hands-on: Send results to Galaxy
- Click on Send results to Galaxy on each query to send them to the last used Galaxy history
- Get back to galaxy and wait for the dataset (reload if needed)
Now that you have used AskOmics to generate this final tabular file, you can continue analysing it with other Galaxy tools. If you are done, don’t forget to close the AskOmics instance by going to the “User” > “Active Interactive Tools” page.
Conclusion
In this tutorial we have seen how to use AskOmics Interactive Tool. We launch the tools with a set of input files, then we have integrated these files into RDF and finally, we built complex queries over this local datasets and neXtProt to answer a biological question.
Key points
AskOmics helps to integrate multiple data sources into an RDF database without prior knowledge in the Semantic Web
It can also be connected to external SPARQL endpoints, such as neXtProt
AskOmics helps to perform complex SPARQL queries without knowing SPARQL, using a user-friendly interface
Frequently Asked Questions
Have questions about this tutorial? Check out the tutorial FAQ page or the FAQ page for the Transcriptomics topic to see if your question is listed there. If not, please ask your question on the GTN Gitter Channel or the Galaxy Help ForumUseful literature
Further information, including links to documentation and original publications, regarding the tools, analysis techniques and the interpretation of results described in this tutorial can be found here.
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Citing this Tutorial
- Xavier Garnier, Anthony Bretaudeau, Anne Siegel, Olivier Dameron, Mateo Boudet, 2022 RNA-Seq analysis with AskOmics Interactive Tool (Galaxy Training Materials). https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/rna-seq-analysis-with-askomics-it/tutorial.html Online; accessed TODAY
- Batut et al., 2018 Community-Driven Data Analysis Training for Biology Cell Systems 10.1016/j.cels.2018.05.012
Congratulations on successfully completing this tutorial!@misc{transcriptomics-rna-seq-analysis-with-askomics-it, author = "Xavier Garnier and Anthony Bretaudeau and Anne Siegel and Olivier Dameron and Mateo Boudet", title = "RNA-Seq analysis with AskOmics Interactive Tool (Galaxy Training Materials)", year = "2022", month = "10", day = "18" url = "\url{https://training.galaxyproject.org/training-material/topics/transcriptomics/tutorials/rna-seq-analysis-with-askomics-it/tutorial.html}", note = "[Online; accessed TODAY]" } @article{Batut_2018, doi = {10.1016/j.cels.2018.05.012}, url = {https://doi.org/10.1016%2Fj.cels.2018.05.012}, year = 2018, month = {jun}, publisher = {Elsevier {BV}}, volume = {6}, number = {6}, pages = {752--758.e1}, author = {B{\'{e}}r{\'{e}}nice Batut and Saskia Hiltemann and Andrea Bagnacani and Dannon Baker and Vivek Bhardwaj and Clemens Blank and Anthony Bretaudeau and Loraine Brillet-Gu{\'{e}}guen and Martin {\v{C}}ech and John Chilton and Dave Clements and Olivia Doppelt-Azeroual and Anika Erxleben and Mallory Ann Freeberg and Simon Gladman and Youri Hoogstrate and Hans-Rudolf Hotz and Torsten Houwaart and Pratik Jagtap and Delphine Larivi{\`{e}}re and Gildas Le Corguill{\'{e}} and Thomas Manke and Fabien Mareuil and Fidel Ram{\'{\i}}rez and Devon Ryan and Florian Christoph Sigloch and Nicola Soranzo and Joachim Wolff and Pavankumar Videm and Markus Wolfien and Aisanjiang Wubuli and Dilmurat Yusuf and James Taylor and Rolf Backofen and Anton Nekrutenko and Björn Grüning}, title = {Community-Driven Data Analysis Training for Biology}, journal = {Cell Systems} }