Wolbachia MLST protocols

Following is a quick guideline of the steps necessary for typing a Wolbachia strain. Please refer to the protocols below for detailed information.

Step flowchart

1. Choose the sample of strains you wish to type.
2. Download the Isolates Form (below), gather all possible information about each strain and start filling the form.
3. For each strain, sequence the five MLST genes using primers and protocol provided below (protocol #1 or #2 for single or double AB infected species, respectively). Sequencing of wsp is not required, but strongly suggested (see the wsp database for primers and protocol).
4. Prepare sequences in FASTA format using the allele templates (below) as reference (for details on this and the following steps please refer to protocol #3 below).
5. Query the Profiles database for allele assignment of all the your MLST sequences.
6. Send all new alleles to the curator (laurab@ucr.edu).
7. Complete the Isolates Form with the allelic profiles.
8. Query each allelic profile to the Profiles database.
9. Download the Allelic profiles form (below) and fill it with the new allelic profiles.
10. Send the following files to the curator (laurab@ucr.edu):
    • Complete Allelic profiles form (containing only the new allelic profiles discovered)
    • Complete Isolates form

The protocols below contain detailed information on the experimental conditions and procedures of the steps listed above.

1. Amplification and sequencing of single infected individuals
2. Amplification and sequencing of double AB infected individuals
3. Preparation and submission of data
4. Allele templates
5. Submission forms