1 Overview

This tutorial describes how to use the Ridom SeqSphere+ software to analyze outbreak strains with cgMLST.

Reference mapped Mycobacterium tuberculosis whole genome shotgun (WGS) data of 4 samples of a community outbreak (isolate1, isolate2_mother_child, isolate3_mother_child, isolate4) is analyzed with the predefined public cgMLST scheme for the M. tuberculosis complex. All 4 samples are part of a larger outbreak consisting of in total 26 isolates and all exhibiting identical IS6110 DNA fingerprint and spoligotyping patterns. The outbreak was published by T. Kohl et al. (JCM 52: 2479, 2014).

M. tuberculosis is used exemplarily for this demonstration. However, by reading this tutorial you should be able to define your own projects for all species with a public available cgMLST scheme in the Task Template Sphere. If you are analyzing a species for which no public cgMLST is available yet, please take a look at Core Genome MLST Schemes.

Pre-assembled WGS data is used for this tutorial. To learn how to assemble WGS data with Ridom SeqSphere+ see the Pipeline Tutorial.

2 Preliminaries

  • Installation: This tutorial requires a running SeqSphere+ client and server. Start the SeqSphere+ server, then start the SeqSphere+ client and initialize the database. For evaluation purpose a free evaluation license can be requested.
The example data contains whole genome shotgun (WGS) data for 4 isolates of Mycobacterium tuberculosis. The isolates are part of retrospective study where isolates of 2150 patients were analyzed with classical genotyping (Hamburg, 15 years). All 4 isolates are identical for a certain IS6110 and Spoligo type (Haarlem lineage). 2 of the 4 isolates have an epidemiological link, the 2 others have no epidemiological link. The WGS data was reference mapped to strain H37Rv (cgMLST reference strain). The data was provided by S. Niemann et al., Research Center Borstel & R. Diel, Univ. Kiel, Germany.

3 Creating Project with DB Scheme and Tasks

  • Step 1: Create a new Project with the menu: File | New | Create Project. A project is typically set-up for samples of the same species. Occasionally for very closely related species, e.g., Mycobacterium tuberculosis complex or Brucella, a project holds samples of several species. For certain applications also sub-species specific, e.g., STEC, projects are recommended.
  • Step 2: Enter a name for your Project (e.g., MTBC outbreak). For the Mycobcaterium tuberculosis complex a special database scheme exists that provides some species specific database fields. Choose Mycobacterium tuberculosis/bovis/africanum in the DB Scheme field.


Seqsphere tutorial mtbc def 01 start.png


  • Step 3: Press Button16-WebDownload.png Download & Add in Task Templates section.
  • Step 4: Choose as organism Mycobcaterium tuberculosis complex.


Seqsphere tutorial mtbc def 02 store.png


  • Step 5: There are two Task Templates available for M. tuberculosis: cgMLST and Accessory. The cgMLST Task Template defines the 2891 genes of the seed genome strain H37Rv that are used for the stable typing and for the definition of the complex type. The Accessory Task Templates defines in addition 755 genes that do not belong to the core genome. However, they can be used to increase the discriminatory power if the resolution of cgMLST is not high enough. Both Task Templates are preselected. Press OK to download and add them to the Project.


Seqsphere tutorial mtbc def 03 project.png


  • Step 6: The Project now contains 2 Task Templates. Confirm with 'OK' to save the Project.
Ridom SeqSphere+ is a resequencing software. Once you have setup a project like this you can literally analyze hundreds/thousands of sequence data automatically.

4 Import Outbreak Strains to cgMLST

  • Step 1: Choose from the menu File | Process Assembled Genome Data


Seqsphere tutorial mtbc import 01 start.png


  • Step 2: The new cgMLST Project you just created and the two Task Templates should be preselected already. Now use the button Open.gif Add from File and choose the 4 fasta-files from the tutorial data folder. Those files are contigs from NGS data that were reference mapped against H37Rv (NC_000962.3) which is the seed genome of the cgMLST scheme. Push the Open button.


Seqsphere tutorial mtbc import 02 select.png


  • Step 3: The upcoming window allows to specify sequencing information. Go with the default settings here by clicking OK. Confirm again with OK in the Process Assembled Genome Data window to start the import process.


Seqsphere tutorial mtbc import 03 seqspec.png


  • Step 4: Ridom SeqSphere+ now loads all input data and searches for the target reference sequences that are defined in the Task Template (using a built-in BLAST).
  • Step 5: Once the import is completed after some minutes, a dialog window shows the imported Samples. Press Load Samples to open the newly processed Samples.


Seqsphere tutorial mtbc import 05 finished.png


  • Step 6: The upcoming navigation tree shows all new Samples. Each Sample node in the navigation has two sub nodes: The cgMLST task and the Accessory task. Below the task nodes there are the target nodes. Each target node represents one sequence (here a gene) extracted from the input data (genomes or wgs contigs). The targets can have different states:
    • Button16-Good Assembly.png Good Targets (green) were extracted and fulfilled all requirements that are defined in the Target QC Procedure of the Task Template.
    • Button16-Failed Assembly.png Failed Targets (red) were extracted, but failed at least in one of the requirements that are defined in those parameters. For example, they may have frame shifts and incorrect lengths compared to the published H37Rv strain sequence that was used as reference.
    • Button16-Missing Assembly.png Not Found Targets (gray) were not extracted (because the match did not reached the thresholds in the previous step)


Seqsphere tutorial mtbc import 05 imported.png


  • Step 7: Select Button16-Crosshairs.png Tools | Position Navigator from the menu. By default all red targets are selected, so just click OK. A confirmation dialog appears. Press OK to continue. After a short while the Position Navigator is opened. Click on the tab Targets with Failed QC Procedure to see a table with the problems that appeared in the imported sequence data. You can double click on a row in the table to jump to the according position in the contig.


Seqsphere tutorial mtbc import 06 posnav.png


5 Phylogentic Analyzing with cgMLST

  • Step 1: Choose from the menu Tools | Comparison Table to perform phylogenetic analysis.


Seqsphere tutorial mtbc phylo 01 newdef.png


  • Step 2: In the Comparison Table dialog go to the first tab "Create New". In the Choose Samples section the new previously created project (e.g., MTBC outbreak). In the Choose Genotypings Schemes section at the bottom cgMLST should already be preselected. Press the Create Comparison Table button to confirm and create the comparison table.


Seqsphere tutorial mtbc phylo 02 comptable1.png


  • Step 3: The comparison table is opened and shows the data for the four Samples. The columns with a green header are used for distance calculation. Some table cells contain missing values. Those appear if a cgMLST target was not found at all in an input sequence ("? (not found)"), or if the Target QC Procedure for this target has failed, e.g., because of a frame shift error ("? (failed)"). The first column in the table shows number of missing values per row.
  • Step 4: Right-click on the column Complex Type (fifth column) and choose from the menu Button16-GroupByValues.png Set Color Groups by Column Values. Leave the upcoming dialog to defaults and confirm with OK. The Sample rows are now colored by the different cgMLST Complex Types.


Seqsphere tutorial mtbc phylo 02 comptable2.png


  • Step 5: Press the Mst.png Minimum Spanning Tree button in the toolbar to calculate the distances between the Samples and draw a minimum spanning tree for them. Because the table contains missing data, it must be confirmed that the missing values are ignored pairwise. Confirm with OK.


Seqsphere tutorial mtbc phylo 03 mst.png


  • Step 6: The minimum spanning tree is calculated for the allelic profiles of the 2,891 cgMLST targets (pairwise ignored missing values) and is shown in a new window. By default the nodes are again colored by the Complex Type (CT) and it can be easily seen that 3 of the 4 isolates have the same Complex Type. Just Isolate4 belongs to a different Complex Type.
Two conclusions can be drawn from this tree.
  • One of the isolates without epidemiological information (Isolate1) has the same Complex Type and a close distance to two other isolates (3-4 alleles) with an epidemiological link (mother/child). This indicates epidemiological relationship.
  • The other isolate without epidemiological information (Isolate4) has a different Complex Type and a distance of 118 alleles to the three other isolates. This clearly excludes recent transmission via one of those three patients.

The MST can be exported by clicking the Button16-Export.gif Export MST icon of the toolbar. In the upcoming Export MST file dialog choose as file type the Scalable Vector Graphics (*.svg) or Windows Enhanced Metafile (*.emf) format. Note that EMF or SVG are vector graphics formats and therefore suited for finishing publication ready figures. EMF files can imported and scaled by MS PowerPoint. SVG files can be edited, e.g., with Adobe Illustrator or the open-source InkScape tool (once the file is loaded first ungroup all objects).

For later usage of the comparison table and the MST a comparison table snaphsot can be stored by using the first button Button16-Snapshot.png in the comparison table or the MST window, or the according function in the File menu.