Table of contents¶

Microscope Platform user documentation.

The MicroScope platform is available at this URL: https://www.genoscope.cns.fr/agc/microscope.

MicroScope Platform Overview¶

Interface¶

Overview¶

Navigation Menu¶

How to use the Main Navigation Menu?¶

Item #1. Login Interface:

Fill the username and password fields as described in the Email sent to you at account creation. After you login, you will have access to all public sequences, as well as private sequences corresponding to your project. Furthermore, you may have annotation rights on certain sequences (if defined in your account parameters).

Tip

Considering the account creation: we will create new accounts only following requests from project leaders. Please ask your project leader to use his own Account & Right Management interface in order to open your account.

Item #2. Reference Sequence selection menu:

From this menu, you can select your Reference Genome/Replicon. Click on the Change button to open a popup organism selection interface, select your reference organism, then click on the Set Selection button. This action will reload the main webpage with the data corresponding to the Organism you selected as reference.

The popup interface will display all the Organisms for which you have, at least, Read rights. This corresponds to Public sequences + Account Restricted sequences.

The select menu below the Change button lists the corresponding organism replicons. Change the selection in this submenu to switch to the replicon you want to explore.

Tip

After logging in, you will have access to the My Favourite Organisms functionality available in the User Panel section. Considering you have registered some favourites in our database with this interface, you’ll note that if you hover your mouse pointer the Change button, a popup will appear. This lists your favourite organism selection. By clicking on one of this organisms, the system will set this one as the new reference organism. This allows a quick access to a personal set of organisms.

Item #3. Navigation Submenu:

During your exploration and annotation work, this menu will indicate your position in the MicroScope’s tools tree, offering users an easy way to locate themselves on the platform.

Item #4. Text Format functionality:

This button will export the displayed web page into a text-converted file easily importable into a spreadsheet like Microsoft Excel or OpenOffice Calc. Click on the button, save the file to your computer, then load it into your preferred spreadsheet program. This file is dynamically created, so you may have to edit (delete) some of the content in order to keep only the data of interest.

Item #5. Help button:

By clicking on this button, you’ll be redirected to the MicroScope Tutorial. You will get a list of help articles related to the tool you’re using at the moment. In case of no correspondences, you’ll be invited to browse the whole content of the tutorial.

Browsing Result Tables¶

How to sort results?¶

Most of result tables provides a default sort (grey-coloured column). To sort results as you wish, simply click on the corresponding column header. Each click will alternate between ASC (ascending order) sort or DESC (descending order) sort. Also, the system provides a multi-sort functionality, to sort and switch on multiple columns. Simply hold your «SHIFT» key and click on column headers you want to multi-sort.

How to filter results?¶

Each result table provides a text area called «Search:» . Enter some characters into this box in order to filter results: each row matching your keywords will be kept, whereas the others will be hidden dynamically.

How to choose the number of results to display per page?¶

Each result table provides a select menu called «Show X Results». Change the value to display the corresponding number of results per page. Values are: 10 (default), 25, 50, 100 or All.

How to export results?¶

Each result table provides buttons called Copy (1) and CSV (2).

Using the Copy button will copy to clipboard each row of your result table in a tab-delimited text format

Using the CSV button will export your result table in a CSV file, fully compatible with spreadsheets like Microsoft Excel, or Open Office Calc

How to print results?¶

Clicking on the Print button will display only the result table within your current window, hiding all the others HTML elements. Then, use your browser’s menu bar to print the displayed table.

Tip

You can leave the «Print Mode» and go back to the original window by clicking your «ESC (Escape)» key.

Annotation¶

In progress

BLAST results¶

What is the meaning of the minLrap and maxLrap values?¶

These values are ratios of alignment lengths computed for each comparison using the BLAST software :

minLrap = Lmatch/min(Lprot1, Lprot2)
maxLrap = Lmatch/max(Lprot1, Lprot2)

where Lmatch = length of the match, Lprot1 = length of protein 1, Lprot2 = length of protein 2.

if minLrap=1 and maxLrap=1 => the 2 proteins both align on their whole length

if minLrap=1 ans maxLrap<1 => one of the proteins is longer than the other, or the alignment is partial. Different interpretations are possible:

the longer protein is a modular protein (domain fusion/fission)
there is an erroneous start codon for one of the 2 genes
the smaller gene is a fragment (pseudogene).
a frameshift (due to a sequencing error or not) causes a premature stop codon in one of the genes.

if minLrap<1 and maxLrap<1 => the sequences are poorly aligned. We can observe this kind of situation in the case of gene remnants.

What is the meaning of orderQ and orderB values?¶

The orderQ and orderB values give an information about the rank of the BLAST hit for a protein of the query genome (orderQ) or for a protein of a databank (orderB).

Best bidirectional Best Hits (BBH) will have a 1:1 relationship The following Best hits will have 1<=>n relationship

Tip

These indicators can be useful to identify fusion/fission events.

Tools¶

Which program is used to detect the repeats ?¶

Repeat detection is performed by the Repsek program.

More: http://wwwabi.snv.jussieu.fr/ public/RepSeek/

Reference: Achaz G, Boyer F, Rocha EP, Viari A, Coissac E. Repseek, a tool to retrieve approximate repeats from large DNA sequences. Bioinformatics. 2007 Jan1;23(1):119-21.

What is Artemis?¶

Artemis is a free genome viewer and annotation tool that allows visualisation of sequence features and the results of sequence analyses. It also supports all six-frame translations. It has been developed at the Sanger Institute.

More: http://www.sanger.ac.uk/resources/software/artemis/

Reference: Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B. Artemis: sequence visualization and annotation. Bioinformatics. 2000 Oct;16(10):944-5

What is the “BioProcess” classification?¶

This functional classification is based on the CMR JCVI Role IDs.

This field is optionally filled in during the expert annotation process.

What is the “Roles” classification?¶

This functional classification corresponds to the MultiFun classification which has been developed by Monica Riley for E. coli (http://genprotec.mbl.edu/).

Reference:Serres MH, Riley M. MultiFun, a multifunctional classification scheme for Escherichia coli K-12 gene products. Microb Comp Genomics. 2000;5(4):205-22.

This field is optionally filled in during the expert annotation process.

What is HAMAP?¶

HAMAP (High-quality Automated and Manual Annotation of microbial Proteomes) is a system, based on manual protein annotation, that identifies and semi-automatically annotates proteins that are part of well-conserved families or subfamilies: the HAMAP families. HAMAP is based on manually created family rules and is applied to bacterial, archaeal and plastid-encoded proteins.

More: http://www.expasy.ch/sprot/hamap/

Reference:

HAMAP: a database of completely sequenced microbial proteome sets and manually curated microbial protein families in UniProtKB/Swiss-Prot. Lima T et al (2009) Nucleic Acids Res. 2009 Jan;37(Database issue):D471-8.

What is UniProt?¶

The Universal Protein Resource (UniProt) is a comprehensive resource for protein sequence and annotation data. The mission of UniProt is to provide the scientific community with a comprehensive, high-quality and freely accessible ressource of protein sequence and functional information.

The UniProt Knowledgebase consists of two sections:

Swiss-Prot which contains high quality manually annotated and non-redundant protein sequences. This database brings together experimental results, computed features and scientific conclusions.
TrEMBL which contains protein sequences associated with computationally generated annotation and large-scale functional characterization that await full manual annotation.

More than 99% of the protein sequences provided by UniProtKB are derived from the translation of the coding sequences (CDS) which have been submitted to the public nucleic acid databases, the EMBL-Bank/GenBank/DDBJ databases. All these sequences, as well as the related data submitted by the authors, are automatically integrated into UniProtKB/TrEMBL.

More: http://www.uniprot.org/

Reference: UniProt Consortium. The Universal Protein Resource (UniProt) in 2010. Nucleic Acids Res. 2010 Jan;38(Database issue):D142-8

What is PRIAM?¶

PRIAM is a method for automated enzyme detection in a fully sequenced genome, based on all sequences available in the ENZYME database (http://www.expasy.ch/enzyme/). PRIAM relies on sets of position-specific score matrices (PSSMs) automatically tailored for each ENZYME entry. The whole Swiss-Prot database has been used to parametrise and to assess the method.

More: http://priam.prabi.fr/

Reference: Clotilde Claudel-Renard, Claude Chevalet, Thomas Faraut and Daniel Kahn / Enzyme-specific profiles for genome annotation: PRIAM Nucleic Acids Research, 2003, Vol. 31, No. 22 6633-6639

What are MetaCyc Pathways?¶

MetaCyc pathways are metabolic networks as define in the MetaCyc Database.

Caspi et al., 2010, “The MetaCyc Database of metabolic pathways and enzymes and the BioCyc collection of Pathway/Genome Databases”, Nucleic Acids Research

The presence or absence of a MetaCyc metabolic pathway is predicted by the Pathway-tools algorithm in this organism.

P. Karp, S. Paley, and P. Romero “The Pathway Tools Software,” Bioinformatics 18:S225-32 2002

What is COGnitor?¶

COGnitor compares a sequence to the COG database by using BLASTP. Clusters of Orthologous Groups of proteins (COGs) were established by comparing protein sequences encoded in complete genomes, representing major phylogenetic lineages. Each COG consists of individual proteins or groups of paralogs from at least 3 lineages and thus corresponds to an ancient conserved domain.

More: http://www.ncbi.nlm.nih.gov/COG/

Reference:

Tatusov RL, Koonin EV, Lipman DJ. A genomic perspective on protein families. Science. 1997 Oct 24;278(5338):631-7.

What is FigFam?¶

“FIGfams, a new collection of over 100 000 protein families that are the product of manual curation and close strain comparison. Using the Subsystem approach the manual curation is carried out, ensuring a previously unattained degree of throughput and consistency. FIGfams are based on over 950 000 manually annotated proteins and across many hundred Bacteria and Archaea. Associated with each FIGfam is a two-tiered, rapid, accurate decision procedure to determine family membership for new proteins. FIGfams are freely available under an open source license.” (quote from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2777423/ )

What is PsortB?¶

PsortB is an open-source tool for protein sub-cellular localization prediction in bacteria.

More: http://www.psort.org/

Reference: Gardy JL et al (2005) PSORTb v.2.0: expanded prediction of bacterial protein subcellular localization and insights gained from comparative proteome analysis. Bioinformatics. Mar1;21(5):617-23. Epub 2004 Oct 22.

What is InterPro?¶

InterPro is an integrated database of predictive protein “signatures” used for the classification and automatic annotation of proteins and genomes. InterPro classifies sequences at superfamily, family and subfamily levels, predicting the occurrence of functional domains, repeats and important sites. InterPro adds in-depth annotation, including GO terms, to the protein signatures.

More: http://www.ebi.ac.uk/interpro/

Reference: Hunter S, et al. InterPro: the integrative protein signature database. Nucleic Acids Res. 2009 Jan;37(Database issue):D211-5. Epub 2008 Oct 21.

What is SignalP ?¶

SignalP (version 4.1) predicts the presence and location of signal peptide cleavage sites in amino acid sequences from different organisms: Gram-positive prokaryotes, Gram-negative prokaryotes, and eukaryotes. The method incorporates a prediction of cleavage sites and a signal peptide/non-signal peptide prediction based on a combination of several artificial neural networks and hidden Markov models.

Reference:

SignalP 4.0: discriminating signal peptides from transmembrane regions. Thomas Nordahl Petersen, Søren Brunak, Gunnar von Heijne & Henrik Nielsen. Nature Methods, 8:785-786, 2011.

What is TMHMM?¶

TMHMM (version 2.0c) is a program for the prediction of transmembrane helices based on a hidden Markov model. The program reads a fasta-formatted protein sequence and predicts locations of transmembrane, intracellular and extracellular regions.

More: http://www.cbs.dtu.dk/services/TMHMM/

References:

Sonnhammer, E., et al. (1998) A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. ISMB, 6, 175-182.

Krogh, A., et al. (2001) Prediction transmembrane protein topology with a hidden markov model: application to complete genomes. J. Mol. Biol., 305, 567-580

What is antiSMASH?¶

antiSMASH allows the rapid genome-wide identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genomes. It integrates and cross-links with a large number of in silico secondary metabolite analysis tools that have been published earlier.

More: http://antismash.secondarymetabolites.org/

References:

Blin, K. et al. (2019) antiSMASH 5.0: updates to the secondary metabolite genome mining pipeline. Nucleic Acids Research, 47, W81–W87.

What is Circular Genome View?¶

CGView is a Java package which allows to produce high quality, zoomable maps of circular genomes. Its primary purpose is to serve as a component of sequence annotation pipelines, as a mean of generating visual output suitable for the web. Starting with information of one genome and the features to visualize, CGView converts the input into a graphical map (PNG, JPG, or Scalable Vector Graphics format) and completes it with labels, a title, legends, and footnotes.

More: http://wishart.biology.ualberta.ca/cgview/index.html

Reference: Stothard P, Wishart DS. Circular genome visualization and exploration using CGView. Bioinformatics. 2005 Feb 15;21(4):537-9

Important

Note that, since version 3.12.2, MicroScope uses a fork of the applet which allows to export images directly from the GUI. The Wishart Research Group is working on a new version of CGView implemented in JavaScript and we are working toward adapting it. The Java version of CGView is no longer under active development and is based on a deprecated technology.

You can use the CG View toolbar to navigate into the circular map.

From left to right, the buttons are:

Zoom out
Zoom in
View entire map
Move counterclockwise
Move clockwise
Show position in the status bar
Show help in the status bar
Export to file

The Legend checkbox allows to show/hide the legend. The Full view labels checkbox allows to show/hide the labels when showing the entire map.

If you click on a gene name/label the corresponding Gene window will be opened giving you access the full annotation of the gene.

Tip

If the application doesn’t work, it means that Java is not installed on your computer (get the latest version of java here)

Tip

You must allow our software to run without certificate by adding https://www.genoscope.cns.fr/ to the exception list. Read this FAQ to know how to proceed.

Technical Requirements¶

A broadband connection to the Internet is required to use the MicroScope platform, although higher-speed connections are preferable.
A minimal screen resolution of 1280x1024 pixels is needed.
Please enable Javascript and Popup windows. This should be enabled by default on your web browser. Else, check your web browser documentation for further information about how to proceed.
Java Web Start is needed for several functionalities.
Supported Browsers: LABGeM team has tested the MicroScope platform with the following browsers:
- Firefox (all platforms) http://www.mozilla-europe.org/fr/firefox/
- Google Chrome (all platforms) http://www.google.com/chrome
- Apple Safari (Mac OSX) http://www.apple.com/fr/safari/

Login¶

How to login?¶

After your account has been created, you will receive an automated message from LABGeM containing the required login information:

Note

Dear annotator,

This is an automated message from LABGeM: your MicroScope account is now fully active.

The Microscope web interface URL is : https://www.genoscope.cns.fr/agc/microscope

Your login : your_username.

Your password : your_password

Please note that login data is confidential. You may not share your account with anyone, or allow anyone other than you personally to access or use your account.

Best regards, LABGeM Team

Use this information in order to login into your account and get access to private sequences and annotation rights.

On the Login Interface of the Navigation Menu (item #1), near the welcome guest message,

fill the username field with your_username
fill the password field with your_password .
then click on the LOGIN button.

Tip

If you already had an active account on the old MaGe version, your username & password for the new interface remain unchanged.
You can login from any window of the MicroScope interface; there’s no need to login from the homepage (or a specific webpage).

Once you’re logged, the Login Interface will be replaced by your Firstname, your Lastname and a LOGOUT button.

On your first login, you’ll be redirected to the Personal Informations Interface where you’ll be prompted to fill in or update required data before using the platform.

Note

For security reasons, as soon as you finished your daily work, do not forget to click on the LOGOUT button in order to close the session and disconnect yourself from our servers.

Why can’t I connect directly to my Project?¶

Our first advice is « DO NOT PANIC! »

The Microscope projects still exists, but now the system is fully transparent for all users. Once connected to your account, you will have access to the full list of Public and Private Sequences according to your Project, and get the annotation rights as defined in your account settings.

You can manage your own set of preferred organisms (for exemple, your Project’s specific organisms) in a Quick Access Menu, by using the My Favourite Organisms.

Latest news¶

How to be advised about MicroScope latest news?¶

As soon as we release a new version of the Platform (new features, improvements), or if LABGeM team needs to communicate some general information about the platform, an article will be added in the «Latest News» panel, available from the platform’s homepage.

Is there «RSS Feeds»?¶

Yes, we provide «RSS Feeds» you can subscribe to by clicking on RSS pictures, available:

in the footer of webpages:

in the «Latest News» panel:

Sequence and Genome selection¶

Since MicroScope version 3.13.0, the selection of sequences and genomes is based on a new selector that has been designed to allow interactive and efficient selection of several sequences or genomes in large lists. It features selection based on several criteria and suggestions.

In this section, selection of Genome means that you are going to select the entire organism including all the replicons. Selection of Sequence means that you are going to select the replicon you want to work on. When talking indistinctly of genome or sequence, we use the term object.

Sequences and genomes come either from MicroScope (PkGDB) or from NCBI RefSeq.

There are two kinds of selectors in the platform (the Simple Selector and the Advanced Selector) which are described in the following sections.

Generally speaking a page use either a simple selector or 1 or 2 advanced ones. For instance, the Keywords Search Tool page use a simple selector in single mode and an advanced selector in multiple mode.

However, some pages use several selectors (of any type), using both PkGDB or NCBI RefSeq. For instance, the Gene phyloprofile page uses 4 advanced selectors (2 from PkGDB and 2 from RefSeq).

Simple Selector¶

This selector is used to select:

a single genome based on the strain name

a single sequence based on the sequence name

It’s similar to the old selector in MicroScope but offers suggestions.

This selector is used in the homepage to select the reference genome and more generally in pages where you must select a reference object (e.g. Lineplot).

It is also used for instance in the following pages:

Pattern Searches (for Sequence Selection)

Genome Browser (for Genome Selection but coupled with a replicon selector)

Note that your favourite organisms will always show up first in this selector.

When the page opens, the selector is displayed like this (it may take some time to load):

Note that the exact appearance of this selector may depend on the page.

Example¶

To select a reference genome on the home page, type in some characters of its strain name. A list of genomes matching this characters will open. From this list, you can select the genome you want.

For example, if you type “escher”, the following list will open:

Note that the search is case-insensitive.

Also you can type any character (not just the beginning). For example, if you type “k12”, the following list will open:

Advanced Selector¶

This selector is used to select one or several objects based on the NCBI taxonomy, strain name or MICGC.

This selector is used for instance in the following pages:

Blast Searches (for Sequence Selection)

Genome Clustering (for Genome Selection)

Gene phyloprofile (for Genome Selection and Sequence Selection)

My Favourite Organisms (for Genome Selection)

Overview¶

When the page opens, the selector is displayed like below (it may take some time to load):

To start selecting organisms click on the Edit button. The selector opens as shown below:

The window is divided in 5 parts:

the Search Criterion and Search Field are used to create filters on the list of objects from the data source; see The search field and the filters for detailed explanation on those fields

the Pre-selection Zone is used to select objects among the filters results

the Selection Zone shows the list of currently selected objects

the Add/Remove buttons allows to transfer objects between the Pre-selection Zone and the Selection Zone

The general usage of the selectors is as follows. You can use the Search Criterion and Search Field to filter the list of all objects from the data source.

Filters can be constructed from:

the Strain name when selecting a genome or the Sequence when selecting a sequence

the Taxonomy of the object (genome or sequence)

the MICGC to which the object belong (see MICGC)

See The search field and the filters for detailled explanation on filters.

The Pre-selection Zone will display the objects that match the filters. You can then select objects from this list and add them to the Selection Zone with the Add Button (green arrow).

If you want to remove objects from the Selection Zone, select them and use the Remove Button (red arrow). See Selection Zone to learn more about the Selection Zone (including the use of filters in it).

You can use the Pre-selection Zone several times with different filters. This allows to create more complex selections.

When satisfied with the list in the Selection Zone, click on Save. The selection window will close and you will return to the page you are interested in for further analysis.

The Reset button will revert both zones (Selection Zone and Pre-selection Zone) to their initial value (i.e. when the page was opened). The selection window stays open so you can restart the selection.

The Cancel button button cancels all the changes done in the current selector (i.e the list of selected organisms is not changed) and closes the selection window.

Example¶

In this example, will we show how to use the advanced selector to select some genomes from the phylum Actinobacteria and whose strain name contains some characters.

If you want to select sequences, the procedure is similar (the main difference being that the Search Criterion contains Sequence and not Strain name).

Select by taxonomy¶

The first step is to filter genomes in the Actinobacteria phylum. To do so, open the selector and select Taxonomy in the Search Criterion. Then type “actinobacteria” in the Search Field. You will notice that suggestions are shown as you are typing.

_images/advanced-selector-select-actinobacteria.png

Filters are shown in the drop down list. In taxonomy mode, filters can operate on any taxonomic level. Click on “Actinobacteria”.

The list of all genomes in the Actinobacteria phylum is now in the Pre-selection Zone.

_images/advanced-selector-list-actinobacteria-genus.png

Note that the filter and the number of genomes filtered appear on the interface. In this example, we have specified the phylum exactly. Hence the filter is “phylum is ‘Actinobacteria’”. See The search field and the filters for more detailled explanations.

By default, genomes are grouped by Genus. Use the “Display by” menu to group by phylum.

_images/advanced-selector-list-actinobacteria-phylum.png

Select by strain name¶

We will now select genomes whose strain name contains “bifi”. To do so, select Strain name in the Search Criterion and type “bifi” in the Search Field.

_images/advanced-selector-select-bifi.png

The list of genomes that match both filters is displayed:

Final selection¶

We can now select some genomes from the filtered list in Pre-selection Zone. To do so, simply select one of them by clicking on it and click on the Add Button.

As you can see, the number of genomes in the Pre-selection Zone is updated. See How to select my organisms of interest? for detailled description.

Congratulations, you have made your first advanced selection in MicroScope ! The rest of this page explains some details about the advanced selector.

Detailed description¶

The search field and the filters¶

The Search Criterion allows to choose on which aspect you want to filter. Typing in the Search Field, will bring suggestions.

Strain name/Sequence filters by name of genome/sequence
Taxonomy filters by taxonomic (NCBI based) information
MICGC filters objects in a MICGC and Tree.

Those suggestions are in fact filters. There are 2 kinds of filters:

partial filter (shown in red in the image below): the genus must contain “Acinetobacter”

exact filter (shown in green in the image below): the genus must be exactly “Acinetobacter”

Pressing enter at any time in the Search Field creates partial filter.

Clicking on a filter will add it.

You can add several filters to improve the accuracy of your pre-selection.

To remove a filter, click on the little “x” next to its name.

How to select my organisms of interest?¶

To select an object, move the mouse with the button down on the wanted genomes in the Pre-selection Zone (shift + click works too). Then press the green button to put them in the Selection Zone.

Tip

You can select the group of genome/sequence by double clicking on the bold tittle inside the Pre-selection Zone.

Selection Zone¶

The Selection Zone is there to allow you to see all the selected object for the analysis. You can remove some of them by moving the mouse with the button down and pressing the red button to remove them from the Selection Zone. If the active filter allow them, they will appear in the Pre-selection Zone.

When you are satisfied with your selection, press the save button to continue the analysis.

What is “Advanced filter”?¶

This part allow you to make filter in the Selection Zone to remove objects more efficiently. It works exactly the same as the first search field.

MaGe¶

Genome Browser¶

Overview of the Genome Browser¶

Organisation of the genomic map¶

The MaGe genome browser is organised into 3 parts:

the upper part of the window details the Coding Sequences (CDSs) that have been predicted for reading frames +1, +2 and +3 in the current region
the middle part indicates the position of RNA objects (rRNA, tRNA, misc_RNA) as well as repeated regions (as turquoise rectangles) if any have been detected
the bottom part of the window shows CDSs that have been predicted for reading frames -1, -2 and -3

The predicted CDSs are indicated by rectangles on each frame.

The blue lines symbolize the coding prediction curve. They increase when coding probability is high and drop when the coding probability is low.

What is the meaning of the Genomic Object color code ?¶

The rectangles symbolising each Genomic Object (CDS, RNA…) follow a color code that corresponds to their annotation status, summarized below:

How to move along the sequence ?¶

You can navigate along the selected sequence by using the grey arrows located on the left and right sides of the genomic map.
You can also enter directly a genomic coordinate and then click on VIEW.
Enter a gene name (e.g. dnaA) or a gene label (e.g., ECK0001) and click on Move to. The map is centered on the requested Genomic object or region.

If several genes have been annotated with the same gene name, the window will move to the first occurrence of these genes on the genome sequence.

What does the right click do ?¶

There is a contextual menu in the genome browser, you may obtain different options depending on your position.

Right click on a genomic object:

Open: open the gene annotation editor
Center: the Genome Browser will be reloaded and centered around the corresponding object.
Zoom: the Genome Browser will be reloaded and centered around the corresponding object and the browser length will be adapted.
Getseq: extract the sequence of the selected object.

Right click on an area:

New: allows you to annotate a new object

Right click on a selected area:

Pathways: match KEGG pathway prediction with objects in the considerate area
New: allows you to annotate a new object

Right click on a synteny:

Open: open the synteny window
GeneInfo: open the gene information page
MoveTo: the Genome Browser will be reloaded and centered around the corresponding object in the new selected sequence and the browser length will be adapted.

Why sometimes is there a dark area ?¶

There are different ways to select a specific gene:

From right click on a gene or synteny and use Center or Zoom option
From result tables:

From the toolbar below the synteny maps:

After a Move To action, the Genome Browser will be reloaded and centered around the corresponding area or gene and the selected area will be highlight.

What is the Matrix ?¶

For a given genome several gene Matrices can be built for gene detection. You can select a given matrix be using the Matrix menu located below the genomic map. Then click on View: the Coding prediction curves are updated.

How to access a gene’s information ?¶

1. Enter a specific gene name or gene label into the right-most edit button below the genomic map, then click on Getinfo (opens an editable Genomic Object annotation window)
1. Click on a gene label in the table annotation editor (read-only window)
1. Click directly on a genomic object in the genomic map (editable annotation window)
1. Right-click on a genomic object in the genomic map then select Open option (editable annotation window)

How to access the annotation history of a genomic object ?¶

Click on the History icon in located the table of genomic objects or in the Gene Annotation Editor window toolbar. The history opens in a new window, allowing you to follow the annotation’s evolution as well as the identity of previous annotators. You can send an email to an annotator by clicking on his/her login name.

How to use the “Export to Gene Cart” button ?¶

The Export to Gene Cart button allows you to export all genomic objects contained in the genomic map to a Gene Cart. If you click on the button, a new window opens, offering the choice of creating a new cart or to selecting a pre-existing cart in which store the data. You can access to your gene carts via the Gene Cart Interface.

Can I create a new genomic object ?¶

The NEW button located below the genomic map allows you to create a new genomic object. If you click on the button, a pop-up will open, you have to choose the type of object you want to create, then the Genomic Object Editor window opens. You have to manually fill in all fields to create your new object. You have to specify its Begin, End, Frame, Mutation, Product, … Then click on SAVE.

Please note that you can’t delete a genomic object from the database.

How to read the table of annotated genomic objects ?¶

Sequence: if you click on the DNA icon, it opens a new window with the sequences (nucleic and protein) of the genomic object
Label: it gives you the label of the genomic object. If you click on it, the Gene Annotation Editor will popup for this Genomic Object
Type: CDS, fCDS, tRNA, rRNA misc_RNA…
Gene: gene name if any
Begin: begin position of the genomic object on the sequence
End: end position of the genomic object on the sequence
Length: length of the genomic object, in nucleotides
Frame: reading frame of the genomic object
Product: description of the gene product of the genomic object
Matrix: reference number for the matrix which has been used to predict the genomic object (see What is the Matrix ?)
Evidence: automatic/validated/artefact // inprogress/finished/curated
AmiGene Status: no/Wrong/New
GC content: GC content of the sequence of the genomic object
GC3 content: GC content on the 3rd position of the codons
CAI: Codon Adaptation Index value
Mw: Molecular weight in Daltons
Pi: Isoelectric point
History: Access to the annotation history of the genomic object

Which program is used to detect the repeats ?¶

Repeat detection is performed by the Repsek program.

More: http://wwwabi.snv.jussieu.fr/ public/RepSeek/

Reference: Achaz G, Boyer F, Rocha EP, Viari A, Coissac E. Repseek, a tool to retrieve approximate repeats from large DNA sequences. Bioinformatics. 2007 Jan1;23(1):119-21.

How to read the Repeat Regions table ?¶

Sequence: Access to the nucleic sequence of the repeat region
Id: Label of the repeat region on the replicon
Begin: Begin of the region
End: End of the region
Comments: Number of repeat units contained in the repeat region

If you click on a repeat region label, you obtain the detailed list of the repeat units contained in the repeat region in a new window.

Sequence: Access to the nucleic sequence of the repeat unit
Id: Label of the repeat unit on the replicon
Type: Type of repeat Direct, Tandem or Overlap
Strand: Location of the repeat unit on the reverse R or direct D strand
Begin1: Begin of the first unit
End1: End of the first unit
Length1: Length of the first unit in bp
Begin2: Begin of the second unit
End2: End of the second unit
Length2: Length of the second unit in bp
Ident%: Identity percentage between the 2 repeat units

Syntenies¶

What is a synteny ?¶

Definitions

Synteny: Orthologous gene set having the same local organization in species A and in species B.
Synton: Maximal set of orthologous gene pairs displaying a conserved organization.
Conserved Organization: Relative location of orthologous genes on compared genomes : permutations - insertions/deletions.

Synteny computation algorithm is relying on 2 kinds of relations:

Inter-genomic : Nature of the relationship (similarity, functional class, etc) and ‘correspondence’ between genes (BBH, 1-n relation)
Intra-genomic : Gene ‘co-localisation’ (with a ‘gap’ parameter).

Correspondence relationships are:

Sequence similarity : BlastP Bidirectional Best Hit OR at least 30% identity on 80% of the shortest sequence (minLrap 0.8)
Co-localization: Gap = 5

What are the different display modes for syntenies vizualisation?¶

Two modes are available for the representation of the syntenies : (1)A representation by pairs of genomes from PkGDB database and from NCBI databank. (2)A representation with species grouped by taxonomy.

How to switch from a mode to another one?¶

The «Switch» button (1), between the genome browser and the synteny maps, allows to change your visualization mode. Also, the «Option» button (2) and «Display preference» interface (3) allow to change:

the vizualisation mode.
the taxon choice for the representation with species grouped by taxonomy (Phylum, Class, Order, Family, Species).
the default organism / taxonomy entries selection, so you can manage your own selections.

How to read the synteny maps with representation by pairs of genomes?¶

The synteny maps are calculated for all pairs of genomes from the PkGDB database (first synteny map) or from the NCBI databank (second map). They represent the distribution of homologs of the current genome in other genomes from these databases. Each row on the map corresponds to one genome replicon (chromosome or plasmid) whose name is indicated on the left. In contrast to the genomic map, there is no scale on the synteny map: a rectangle has the same size as the CDS to which it is homolog.

The color of the rectangles reflect illustrate synteny conservation, to the exception of the white color. Thus, a group of rectangles which share a common color shows that there is a conservation of the synteny between the current genome and the genome of the synteny map. Rectangles filled with white indicate homologs that don’t belong to a synteny group. The synteny maps should be read linearly: the color code has to be interpreted by replicon, i.e. by row. The same color on 2 synteny map rows doesn’t indicate any synteny relationship.

When you hover the mouse pointer over a synteny gene, a short summary appears : it indicates the gene label of the homolog, as well as its gene name and product description. It also gives the identity (Id) conservation between the sequence and its homolog on the studied genome. The minLRap and maxLrap values give some indications about the alignment of the 2 proteins.

The filling of a rectangle reflects the alignment quality between the 2 proteins.

Example:

How to read the synteny maps with representation grouped by taxonomy ?¶

Syntenies are computed from the PkGDB database for the first map and from the NCBI databank for the second map. Each line refers to a taxon for which the name is displayed on the left side, followed by the number of different species organized in synteny in the observed genomic region. The taxonomic rank can be modified through the «Option» button.

On the maps, a coloured box represents the synteny conservation with the reference gene for at least an organism of taxon of the row. Boxes have the same size that the corresponding reference gene and the synteny map is lined with Genome Browser to ease comparisons.

The color of the block corresponds to species percentage which have a synteny with the reference gene. This percentage is computed by dividing the organisms number of taxon in synteny for the corresponding gene by the total organisms number of the taxon.

Percentage of species in synteny

How to zoom in on a synteny group ?¶

If you click on a synteny group, it opens a popup synton visualization window which shows a more detailed view of the syntenies.

Representation by pairs of genomes

Representation with species grouped by taxonomy

Artemis¶

What is Artemis?¶

Artemis is a free genome viewer and annotation tool that allows visualisation of sequence features and the results of sequence analyses. It also supports all six-frame translations. It has been developed at the Sanger Institute.

More: http://www.sanger.ac.uk/resources/software/artemis/

Reference: Rutherford K, Parkhill J, Crook J, Horsnell T, Rice P, Rajandream MA, Barrell B. Artemis: sequence visualization and annotation. Bioinformatics. 2000 Oct;16(10):944-5

How to open Artemis ?¶

You can access the Artemis application by using:

Artemis region: the sequence is loaded into Artemis but only the features corresponding to the Genomic objects located in the region which is visualized in the Genome Browser are loaded.
Artemis whole genome: the sequence is loaded into Artemis and all genome features are loaded.

A new window appears with the Artemis interface. All genomic objects are listed in the bottom part of the window using their labels. You can click on the right button of your mouse and select Show Gene names to identify the objects by their gene names instead.

How to use Artemis to identify alternative Start codons ?¶

Double click on an object to select it in the upper part of the window. The object is then positioned at its start position.

Keyboard shortcuts:

ctrl + Y key: Artemis will propose the next possible Start position for your CDS. You can do this several times.
ctrl + U key: Undo your last action.
ctrl + Q key: Select the whole ORF.

Once you have identified an alternative Start codon, you can copy its position and change the value in the Gene annotation editor window of your gene.

What do I do if java doesn’t work on my computer ?¶

Go to the Artemis Website: http://www.sanger.ac.uk/resources/software/artemis/

Download Artemis and install it on your personal computer.

Use the Export functionality to export your genome as an EMBL file. You can then open it with your personal version of Artemis.

Gene annotation editor¶

Overview of the annotation editor¶

How to access to the Gene Annotation Editor?¶

There are two ways of accessing the Gene Annotation Editor:

click on a genomic object on the genomic map
click on a label in the table of genomic objects which is below the genomic map

Important

requesting information via the GetInfo button only calls up a read-only Gene Annotation Editor window.

Overview of the Gene Annotation Editor¶

The Gene Annotation Editor window is made of 4 sections:

a toolbar that allows access to different functionalities
the current annotation of the genomic object. This section can be modified by the annotator (with sufficient rights).
the primary annotation of the genomic object. It corresponds to the MicroScope pipeline automatic annotation (if it is a first annotation) or to the databank annotation (if it is a reannotation project).
the Method results section. This section gives an access to the results obtained by the different tools used for the syntactic and functional annotation process.

Expert annotation of gene function¶

How to fill the Gene Annotation form?¶

As shown in the figure below, not all fields can be modified by the annotator. Furthermore, some of them are required and other are optional. These fields have to be filled after the careful analysis of the different methods results. If your are working on other object than CDS, you may have a different form, if a required field for CDS appear in your form, it’s still required.

Tip

If one of the required field is missing or wrongly filled a warning will appear in the window.

What are the different annotation “Status”?¶

inProgress : the annotator has not finished the expert annotation
finished : the annotator has finished the expert annotation
Curated : the expert annotation has been reviewed by a specialist of the functional process in which the CDS product is involved
Artefact : An artefactual CDS corresponds to a false prediction by the gene detection program. An artefactual CDS should never be similar to any proteins from the databanks (except if the same erroneous annotation has been made in another genomes)
chkSeq : this status is used by the annotator to flag potential sequencing errors in the sequence. When the sequencing is performed at Genoscope, these chkSeq sequences will be sent to the people working in the finishing team. They will then check the assembly to see if the sequence quality is good or not. If needed they can perform some additional PCRs to enhance the data.
chkStart : the annotator suspects that a start position readjustment might be needed for the CDS, but hasn’t done it yet.

How to identify artefacts?¶

How to fill the “Mutation” field?¶

no => Normal CDS
frameshift => CDS for which a true frame-shift has been biologically demonstrated
pseudo => the CDS is part of a pseudogene
partial => the CDS is a gene fragment
gene remnant => the CDS is a highly degraded gene fragment
selenocysteine => the CDS contains a Selenocysteine in its sequence
pyrrolysine => the CDS contains a pyrrolysine in its sequence

How to use the “MetaCyc reaction” field?¶

This field allows user to link one ore more metabolic reactions from MetaCyc (BioCyc) to the current edited gene.

a: Reactions presented at the top of the field have been manually curated by an annotator.
b: A multiple selection list gives quick access to all predicted (unselected) or curated (selected) reactions linked to this gene.
c: A search box allows one to quickly access MetaCyc reactions corresponding to either EC numbers from previous EC number field or a given keyword.

Search box :

Clicking on the “EC” button will search all MetaCyc reactions corresponding to the EC number from the “EC number” field.

The keyword search will look for all MetaCyc reactions having an identifier, a name or involving a compound similar to the given keyword.

Search result :

The search returns a list of MetaCyc reactions, with :

the reaction identifier and name. Identifier is clickable and open the BioCyc reaction card.

And in some cases :

Genes of the organism already linked to this reaction (eg. first row of the example). Genes are flagged with :
- “validated” : reaction has been manually linked to this gene by users.
- “annotated” : reaction has been linked to homologous gene and transferred here from a close genome.
- “predicted” : reaction has been linked to this gene by the pathway-tools algorithm.
If the reaction has no known coding genes but belongs to a pathway predicted to exist in the current organism, a clickable link to the MetaCyc pathway description is given (eg. fourth row of the example).

The “Reset” button deletes all results.

How to use the “Rhea reaction” field?¶

This field allows user to link one ore more metabolic reactions from Rhea to the current edited gene.

a: Reactions presented at the top of the field have been manually curated by an annotator.
b: A multiple selection list gives quick access to all curated reactions linked to this gene.
c: A search box allows one to quickly access Rhea reactions corresponding to either EC numbers from previous EC number field or a given keyword.

Search box :

Clicking on the “EC” button will search all Rhea reactions corresponding to the EC number from the “EC number” field.

The keyword search will look for all Rhea reactions having an identifier, a name, involving a compound name or Chebi identifier similar to the given keyword.

Search result :

Rhea reactions are present in 4 exemplary according to the direction :

bidirectional : <=>
left to right : =>
right to left : <=
*unknown (master reaction) : <?>

The search returns a list of Rhea reactions, with :

the reaction identifier and name. Identifier is clickable and open the Rhea reaction card. By default, the master reaction is presented. Select the direction wanted in the “direction-select”.

And in some cases :

Genes of the organism already linked to this reaction (eg. first row of the example). Genes are flagged with :
- “validated” : reaction has been manually linked to this gene by users.

The “Reset” button deletes all results

How to link a new reaction :

For each reaction in the result set, check-box allows to add a reaction from the result set to the selected element. All reactions selected in the multiple selection list will be saved as validated and linked to this gene. Unselecting a reaction in this list will remove this link from the curated data.

What is the “BioProcess” classification?¶

This functional classification is based on the CMR JCVI Role IDs.

This field is optionally filled in during the expert annotation process.

What is the “Roles” classification?¶

This functional classification corresponds to the MultiFun classification which has been developed by Monica Riley for E. coli.

Reference:Serres MH, Riley M. MultiFun, a multifunctional classification scheme for Escherichia coli K-12 gene products. Microb Comp Genomics. 2000;5(4):205-22.

This field is optionally filled in during the expert annotation process.

How to use the “PubMedID” field?¶

The PubMedID or PMID correspond to the index of a publication on the PubMed section of the NCBI website. You can fill this field when you want to link a publication to your annotation. If you want to enter several publications, you simply have to write the PMIDs separated by commas.

You will find the PMID of a publication directly on Pubmed as shown on the figure below. You can also find PMIDs in the “References” section of the UniProt entries.

If this field is filled you will have a direct access to the publications on PubMed by clicking on the PubMed button on top of the Gene annotation editor window.

How to use the “Additional data” field?¶

The Comments field is dedicated to the annotators who want to leave some notes for themselves or for others annotators from the project.

How to use the “Class” field?¶

The Class annotation categories are useful for assigning a “confidence level” to each gene annotation. It has been inspired by the “protein name confidence” defined in PseudoCAP (Pseudomonas aeruginosa community annotation project).

This information is not given by the automatic functional annotation procedure, except in case of functional annotation transfer from a genome being annotated with MaGe.

The different classes are:

1a : Function from experimental evidences in the studied strain
1b : Function from experimental evidences in the studied species
1c : Function from experimental evidences in the studied genus
2a : Function from experimental evidences in other organisms
2b : Function from indirect experimental evidences (e.g. phenotypes)
3 : Putative function from multiple computational evidences
4 : Unknown function but conserved in other organisms
5 : Unknown function

How to choose the “Class” annotation category?¶

Annotation Rules¶

Considering the Class field, here are some basic annotation rules:

1 a/b/c: Function from experimental evidences in the studied organism/species/genus¶

Gene [optional]
Synonyms [optional]
Product [known]
EC number [optional]
MetaCyc Reaction [optional]
PubMedId [known]
ProductType [known]
Localization [optional]
BioProcess [optional]
Roles [optional]

2a : Function from experimental evidences in other organism¶

Gene [optional]
Synomyms [optional]
Product [known]
EC number [optional]
MetaCyc Reaction [optional]
PubMedId [known]
ProductType [known]
Localization [optional]
BioProcess [optional]
Roles [optional]

2b : Function from indirect experimental evidences (e.g. phenotypes)¶

Gene [optional]
Synonyms [optional]
Product [known]
EC number [optional]
MetaCyc Reaction [optional]
PubMedId [optional]
ProductType [known]
Localization [optional]
BioProcess [optional]
Roles [optional]

3 : Putative function from multiple computational evidences¶

Gene [not allowed]
Synonyms [not allowed]
Product [putative function]:
EC number [optional]
MetaCyc Reaction [optional]
PubMedId [optional]
ProductType [known]
Localization [optional]
BioProcess [optional]
Roles [optional]

4 : Unknown function but conserved in other organisms¶

Gene [not allowed]
Synonyms [not allowed]
Product [conserved … protein of unknown function … ]
EC number [not allowed]
MetaCyc Reaction [optional]
PubMedId [optional]
ProductType [u : unknown]
Localization [optional]
BioProcess [optional]
Roles [optional]

5 : Unknown function¶

Gene [not allowed]
Synonyms [not allowed]
Product [protein of unknown function]
EC number [not allowed]
MetaCyc Reaction [optional]
PubMedId [optional]
ProductType [u : unknown]
Localization [optional]
BioProcess [optional]
Roles [optional]

Start¶

In progress

This menu gives the beginning and the end of the gene sequence according to different softwares. If the indicated start and stops seems to be wrong when compared to those given by the softwares, you can correct them by using Artemis (see Artemis).

Strand: indicates if the CDS is on the direct strand (D) or on the reverse strand (R)
Begin: give the leftmost beginning of the CDS according to the expert or automatic annotations
End: give the ending of the CDS according to the expert or automatic annotations
AMIGene Start: gives the start according to AMIGene
AMIGene Lpcod: gives the coding probability on the length End-Begin +1 according to AMIGene
AMIGene Apcod: gives the length End-AMstart +1 according to AMIGene
Matrix: gives the matrix number (see here)
SHOW Begin: gives the position of the first nucelic acid of the CDS according to SHOW
SHOW End: gives the position of the last nucelic acid of the CDS according to SHOW
SHOW Proba : gives the coding probability on the lenght End-SHOW begin +1 according to SHOW
Prodigal Begin: give the beginning of the CDS according to the expert or automatic annotation
Prodigal End: give the ending of the CDS according to the expert or automatic annotation

Compositional features¶

Gene compositional features¶

This section gives the different compositional features of the studied gene, determined by GenProtFeat.

GC Content:
GC1 Content:
GC2 Content:
GC3 Content:
CAI:
GCskew:
R/Y ratio:

Protein compositional features¶

This section gives the different compositional features of the studied gene, determined by GenProtFeat.

Mw (Da): gives the molecular weight of the protein (Da)
Hydrophobicity:
Tiny:
Small:
Aliphatic:
Aromatic:
NonPolar:
Polar:
Charged:
Basic:
Acidic:
PI: gives the value of the protein isoelectric point
Oxyphobic Index:

Duplications¶

This dataset contains the list of genes of the genome that have an identity > 25% with a minLRap > 0.75 to the selected gene.