Process Genomes with Seed Only or Limited Number of Queries Scheme

• Step 1: Invoke menu function Tools | cgMLST Target Definer and create a cgMLST scheme using only a seed genome if making a soft defined scheme only (J Clin Microbiol. 2017, 55:2086-97) or in case of a stepwise approach, i.e., first hard and then soft defined (J Clin Microbiol. 2024, 62:e01725-23), a seed genome and a very limited number of query genomes. When finished, create a new project that contains only the cgMLST task template.

• Step 2: Process genome data of all genomes that will be used for the soft definition of the cgMLST scheme into the new project.

• Step 3: Delete insufficient genomes or mark them with a tag.

Find Targets to Exclude from Seed Only or Limited Number of Queries Scheme

Warning: For a large number of genomes this process requires a large amount of RAM (around 10 GB per 1000 genomes).

• Step 1: Invoke in menu Tools | Sample QC Overview.

• Step 2: Choose the project from above (probably already preselected).

• Step 3: Click the button Advanced, and then Perform Target Level QC (...).

• Step 4: In the upcoming dialog select in the lower right box the option Include Statistics per Target.

• Step 5: If necessary, define other criteria for the samples, e.g. not containing a tag that marks insufficient data.

• Step 6: Press OK to continue. In the upcoming confirmation dialog proceed with button All Targets.

• Step 7: A confirmation dialog with the pre-selected cgMLST task template will be shown. Confirm with OK to start the calculation.

• Step 8: After statistic calculation has finished and results are shown.

• Step 9: Go to tab 'Samples' > 'Summary' and check the Mean perc. good targets.

• Step 10: Now go to tab 'Targets' > 'Targets'

• Step 9: Sort by Perc. Good in Samples (descending)

• Step 10: Select all rows above a threshold in Perc. Good in Samples (e.g., targets found in 97% or 95% of all samples).

• Step 11: Right-click on the first column Target and invoke Copy Selected Values of Column to Clipboard. It may also be useful to export the sorted table for later use.

Exclude Found Targets from Final cgMLST Scheme

Important: Requires that the list of selected targets for the finally chosen threshold are in the clipboard.

• Step 1: Invoke in menu Tools | cgMLST Target Definer again.

• Step 2: Choose again the same seed genome and if following a stepwise approach also the query genomes.

• Step 3: Go to tab Manually exclude genes and press Add Targets to Exclude

• Step 4: In the upcoming dialog paste into Filter the copied list of selected targets again.

• Step 5: Press Select All.

• Step 6: Clear the filter by pressing the button right of the filter text field.

• Step 7: Press Invert Selection.

• Step 8: Confirm with Move Targets to Accessory.

• Step 9: Enter a descriptive reason for the exclusion of those genes for documentation.

• Step 10: Press the Start button to start the core genome definer process.