1 Overview

This tutorial describes how to use the Ridom Typer software to analyze single target Sanger sequence data (e.g., chromatogram files) with a predefined 16S rDNA reference library.

2 Preliminaries

• This tutorial requires a running Ridom Typer client and server. Start the Ridom Typer server, then start the Ridom Typer client and initialize the database. For evaluation purpose a free evaluation license can be requested.

• This tutorial uses a 16S task template that is not available via the Task Template Sphere. Therefore, download the 16S LTP task template manually as file, the import is described later in this tutorial. The task template is based on the All-Species Living Tree Project (LTP).

Citation: Ludwig W, Viver T, Westram R, Francisco Gago J, Bustos-Caparros E, Knittel K, Amann R, and Rossello-Mora R. Release LTP_12_2020, featuring a new ARB alignment and improved 16S rRNA tree for prokaryotic type strains. Syst Appl Microbiol. 2021, 44: 126218 [PubMed 34111737]

• Download and extract the example data archive Ridom_Typer_Examples_Sanger_16S.zip

3 Downloading Task Template and Creating Project

• Step 1: Create a new Project for use with your sample data with the menu command File | New | Create Project

 

 

• Step 2: Enter a name in the field Project Name (e.g., 16S Tutorial ). The fields Category and Acronym can be left empty.

 

 

• Step 3: Now press Add in Task Templates section to add a task template to the project. The dialog window Add Task Template to Project opens and shows the Task Templates that already exist in the database. Press the button Import from File and choose the downloaded task template file 16S_LTP.tasktemplate

 

 

• Step 4: An editor window opens and shows the 16S LTP task template that will be imported. For the tutorial no further editing is required. Press OK to confirm the dialog and import the task template. The newly imported task template is automatically selected in the Task Templates window. Press again OK to confirm the dialog.

 

 

• Step 5: The imported task template was added to the project. Confirm the dialog with OK to store the new project.

Ridom Typer is a resequencing software. Once you have setup a project like this you can literally analyze hundreds/thousands of sequence data.

4 Assembling and Analyzing Sanger Sequence Data

• Step 1: Choose from the menu File | Process Sanger Sequencing Data. Press the button above the file browser panel on the left, and choose the directory where you extracted the tutorial example data.

 

 

• Step 2: Select the tutorial example data directory or all of the files in it, and press the button in the toolbar in the center of the dialog.

 

 

• Step 3: A dialog opens up to sort the chromatograms (fragments) into Samples. Be sure that the correct 16S project is selected. Select Match by default File Naming. As the file naming is recognized automatically, the six chromatograms will be sorted into three different samples. If this does not work for your own data later, use the button Edit File Naming to adjust the settings (alternatively, a file naming can be defined in the task template). Now confirm the dialog with OK.

 

 

• Step 4: The preview on the right shows the six chromatograms sorted into the three newly defined Samples. Confirm the dialog by pressing the OK button to start assembling the chromatograms and processing the Samples.

 

 

• Step 5: After a short while, the reads are assembled and the three new Samples are listed in the navigation tree on the left of the main window. On the right an overview and the epi metadata of the first Sample is shown, for the processed 16S target a link to the Target and to the Contig are shown. Click on the link Contig to show the assembled sequence data.

 

 

• Step 6: The assembled chromatograms and the contig alignment with the best matching reference sequence from the 16S library is shown. If you click at a position in the Target Sequence section, the corresponding base calls are highlighted in the chromatogram. If necessary, base call errors can be edited manually by right-clicking into the chromatograms. This is not needed for the tutorial sequence data.

For use with further web services (e.g., NCBI Blast search), the consensus can be exported to the clipboard or to a FASTA file by right-clicking on the consensus in the 'Target Sequence' section and choosing Copy Bases of Consensus Area(s) to Clipboard or Export Bases of Consensus Area(s) to File.

In the navigation tree on the right of the window now double-click on the subnode 16S LTP of the first sample. to show the result of the 16S task.

 

 

• Step 7: The BLAST search result is shown in the table. Acinetobacter baumannii is shown in the top of the list. The hits for this 16S task are not filled automatically into the result fields, but they can be inserted manually. For this result, the genus Acinetobacter is clearly detected. Therefore, write Acinetobacter into the field Genus left of the table to define the result.

 

 

5 Storing and Retrieving Samples

• Step 1: Choose from the menu File | Save All to store the samples to the database on your Ridom Typer server. Then choose File | Close All to remove them from the workspace.

• Step 2: Choose File | Search Samples. Select the project in the Project box, and choose 1 day(s) for Recently modified. Then press the Search button.

 

 

• Step 4: The Samples that just have been saved are listed as search result. Close this dialog, and invoke in the menu Tools | Comparison Table.

• Step 5: The new 16S project and the 16S LTP task are pre-selected by default. Confirm the dialog with the Create Comparison Table button.

 

 

• Step 6: A comparison table is shown for the three samples. The column "Genus" of the 16S LTP task contains the value that was entered before.