Using the RAST prokaryotic genome annotation server

RAST is designed to rapidly call and annotate the genes of a complete or essentially complete prokaryotic genome.
RAST, Rapid Annotations based on Subsystem Technology, uses a "Highest Confidence First" assignment
propagation strategy based on manually curated subsystems and subsystem-based protein families that
automatically guarantees a high degree of assignment consistency. RAST returns an analysis of the genes and
subsystems in your genome, as supported by comparative and other forms of evidence.
Because NMPDR and the SEED provide access to all essentially complete, public genomes without a user account,
the use of RAST without an account makes no sense—you must have a free account in order for access to your data
to be kept under your control. The tools available in RAST for comparing your new private data to public genomes
are mostly the same as those available for analyzing public genomes at NMPDR (www.nmpdr.org).
The tour of the site will follow the workflow listed below. For short answers to specific questions, see the RAST
FAQ.

Upload and manage your job
Sequence format and upload steps
Log in and select "Upload New Job" from the "Your Jobs" menu.

Step 1: Browse for the sequence file which must be a plain text file in either FASTA format or GenBank
format only.
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Multiple contigs or replicons of the same genome should all be together in one file.

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Files encoded as html, pdf, rtf, doc, docx, embl, gff3, or gtf will be rejected. Sequences in the
correct FASTA or Genbank format must NOT be in a Microsoft Word document--save as a plain
text (*.txt) file, text encoding Windows (default); do NOT insert line breaks or allow character
sustitution.

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Click the button to "Upload and go to step 2."

Step 2: Provide the name of the organism and choose a translation table.
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If you know or find the taxonomy ID from NCBI, paste it into the text box. Then, when you
select either Bacteria or Archaea with the radio button, the corresponding genus, species, and
strain will autofill accurately. If you do not know or cannot find an ID in the NCBI Taxonomy
database, then fill in the genus (one word), species (one word), and strain (any number of

Your annotation job could be complete in as few as 8 hours. o Select whether the translated proteins should reflect corrected frame shifts if you have low-quality sequence data. Since there are no gene calls in a FASTA file. . • Track progress: All of your jobs are displayed in a table with active headers. • Step 3: Provide information about the sequence data and select settings. o An overview of the progress is shown in the table as a series of colored boxes. o Look at the information in the "Upload summary" tab to confirm that the system detected the sequence data you intended to upload. o Most bacteria use version 11 of the genetic code. o Click the button to "Finish Upload. RAST will provide a dummy ID number corresponding to nothing in the taxonomy database. you may elect to preserve gene calls. • Access completed job: From the details page. you may view or download the annotated genome." Manage your job From the "Your Jobs" menu. this choice would be unavailable.words). you will be directed to your jobs overview upon logging back in. Select the link to view details of one job. but mycoplasmas and spiroplasmas use version 4." If you have logged out. o If you uploaded a GenBank file. select "Jobs Overview. o How to navigate the genome viewer will be discussed below.

the number of genes that are assigned to complete subsystems. request a new group by emailing rast @ mcs. and EMBL. with and without EC numbers. GFF3. Share your annotated genome with one or more other users. • Delete job: First you must click on the "view details" link in the jobs table. . View your annotated genome results Organism Overview page • From the jobs table. click on "view details" and then "Browse annotated genome in SEED Viewer" • The overview page opens with a table that lists how many contigs. o If you would like to share with many people." An intermediate screen will appear to confirm whether you are sure you want to delete. how many genes. if needed.anl. which is accessed by clicking on the pair of people at the far right of the green menu bar . and the subsystem categories represented in your genome. e. o You can share this job with others by clicking the link and adding the email addresses (one at a time) of registered users to whom you would like to grant access to your otherwise private data. Group memberships may be viewed from the account management page. The only action to choose from this menu is "Delete this job. Click the button to do so. a class.g. Then. the green menu bar in the header of the page will provide an option labeled with your job number.o • Download formats include GenBank.gov. This is also where you can change your password.

• To expand the graphic comparison to more genomes. click the advanced button. type in a larger number of genomes (20). select PCH pin for clustered genes. The table is resortable and downloadable. • In the menu bar. then click the button to redraw the graphic. under Organism. Click on any feature ID to open the Annotation Overview page for a selected feature.• The green "Features in Subsystems" tab displays all genes and other features that are automatically included in subsystems because one similar sequence was found for all roles in a functional variant of the subsystem. . • Sets of homologous proteins located in the genomic region are presented in the same color with a numerical label. Annotation Overview pages for individual features • Compare Regions displays the new genome at the top in comparison with 4 other closely related genomes. • All information in the graphic is presented in a table in the next tab. the feature table will display all annotated features in your genome— both those in and not in complete subsystems. select the option to collapse close genomes. or leave at similarity for isolated genes.

Click the "Show" button in the Region column to focus the browser graphic on the selected feature. Click navigation arrows to move forward or back along the genome. • Click on any feature ID in the table to open its Annotation Overview page. you can select the "Carbohydrates" category from the column header. Clicking a feature arrow in the graphic will center the graphic on that protein and color the focus protein red. • The genome browser provides a visual tour of the annotated features. You may choose a larger window. click to open the Glycolysis and Gluconeogenesis subsystem.Walk the chromosome or contigs of your new genome • Browse genome Access to the genome browser is available from the menu bar. and in the hint box at the top of the Organism Overview page. Metabolic reconstruction of your new genome--automated subsystems assignments • The genome overview page displays a pie chart of complete subsystems identified in the genome. Mouse over any feature to pop up its identity. Click on the details button in the focus tab to open the Annotation Overview page for the protein of focus. along with the number or proteins assigned to each. • The table (click the green "Features in Subsystems" tab) provides similar access. under Organism -> Genome Browser. . • The table beneath the graphic allows you to scan or search for a feature of interest. • From the Carbohydrates category either in the chart or table. and you may color the features by subsystem or clustering. • Expand the categories to see subcategories and subsystem names.

The spreadsheet is arranged with functional roles in columns. • Click on any of the genes in the newly annotated genome to open its Annotation Overview page. the new genome is highlighted and displayed in the context of closely related public genomes. genomes in rows. which will open in a new window or tab. . Within one row.• In the subsystem spreadsheet. and genes annotated to those roles in the respective cells. genes that are clustered on the genome are shown in the same color.

then select "Sequencebased comparison" from the Comparative Tools menu. If your private sequence is in many contigs. . go back on your browser to return to the overview. • Step 2: Select up to three comparison genomes. the best selection may be a known. • Step 3: Click button to compute. high-quality genome.How does your genome sequence compare with others? Run the sequence-based comparison tool From the page showing the subsystem. • Step 1: Select a reference genome.

zooepidemicus MGCS10565 genome in order to compare the preserved. • Results are presented in a color-coded table and in a circular map. which allows you to select from all your annotation jobs. • Comparison proteins are listed with their contig number. in order of the contigs/genes in the reference genome. If your private genomes are in multiple contigs. • The example illustrated below uses two private jobs. published gene calls to the RAST-computed gene calls. • The amino acid identity of the comparison genomes relative to the reference is color-coded on a scale that is not linear. • Results are computed on demand in real time using BLASTP to compare every protein in the reference genome to every protein in the comparison genomes. The second two genomes are nephritogenic strains of Streptococcus pyogenes from the list of public genomes. Protein sequence similarities are computed on demand. but logarithmic. and you use a closed. gene number.Result • This tool allows you to select from all your private genomes as well as all public genomes. Notice that the most similar sequences are ribosomal proteins. these results will help order your contigs. . The gene numbers are linked to pop-up boxes that list the annotation (name) as well as the proportion of identical amino acids. the GenBank and FASTA versions of the same Streptococcus equi subsp. and length. and that follows the order of the visible spectrum. public genome as the reference.

select "Function-based comparison. • The table is sortable. this comparison of which functions are annotated in B but not automatically associated with subsystems in A . When genome B is very closely related to the new genome. • Step 3: Click the "Select" button Result • The table opens with all features in your genome (A) or the comparison genome (B) that are associated with a complete subsystem. but not B. in both A and B." • Step 1: Your newly annotated genome is already input as the reference. • Step 2: Highlight one comparison genome in the list. • The first column may be reset to show features associated with subsystems in genome A. or in B but not A. and downloadable. A.How does your genome content compare with others in the database? Run the function-based comparison tool From the Comparative Tools menu of the organism summary page. searchable by subsystem category or name.

• Select those comparisons that require calculation. the similarities between two or more private genomes are calculated only upon request.indicates a place to begin looking at the annotations to evaluate accuracy or find missing functions. Annotations are assigned based on sequence similarity. then click the button to request computation. while inclusion in subsystems is based on the annotation matching that of a functional role of a subsystem. • You will recieve an email when the computation is complete. In order for two or more of your jobs to be displayed in compare regions or other comparative tools. How to include two or more jobs (private organisms) in comparative analyses Set private organism preferences For each individual job. In order to maintain privacy of the data in each job. the protein in your new genome will not be included in the subsystem. if the best sequence match to your new protein has a functional annotation that does not match exactly to the name of a role in a subsystem. It is important to keep in mind that automated subsystem assignments are made only if all roles required for one functional variant of a subsystem have been correctly annotated. • Click the button to check requirements for computation. • Select "Private Organism Preferences" from the Your Jobs menu • Shift the genomes you want to compare with each other into the peers box at right by selecting them (one at a time) and clicking the right-pointing arrow. RAST calculates similarities between all features in the input genome (private genome) and all features the SEED database (public genomes). the similarities of features in the private genomes to each other must be calculated. Therefore. . Such an event may occur when the protein with the best sequence match in the database has not yet been reviewed and anotated by the curator of the subsystem in question.

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