Expand PubMLST Databases Downloads BIGSdb Contact Account

Wolbachia WSP typing protocol

Use the following protocol for amplification and sequencing of wsp in host species carrying a single strain infection. Given the variability of wsp (Baldo et al. 2005), wsp can also be used to detect double/multiple infections by direct sequencing of PCR product. Multiple peaks in a chromatogram will reveal potential multiple infections.

Primers

wsp_F1: GTCCAATARSTGATGARGAAAC
wsp_R1: CYGCACCAAYAGYRCTRTAAA

product size: 603 bp

PCR protocol

This protocol is the same used for amplification of the MLST genes.

Perform PCR reactions in a 40 μL final volume using 2 μL DNA, H2O to volume and reagents at the following concentrations:

ReagentsPCR Final Concentration
Buffer1x
dNTPs0.2 mM
MgCl21.5 mM
Primer F1 μM
Primer R1 μM
Taq DNA polymerase0.5 U

PCR cycling conditions:

After cycling, check that amplification was successful by running 5 μl of each reaction on an agarose gel, with size standards.

Purify the remaining 35 μl and sequence.

Templates (from wMel):

>wsp
ACAAAAGTTGATGGTATTACCTATAAGAAAGACAAGAGTGATTACAGTCCATTAAAACCATCTTTTATAGC
TGGTGGTGGTGCATTTGGTTACAAAATGGACGACATCAGGGTTGATGTTGAAGGAGTTTATTCATACCTAA
ACAAAAATGATGTTAAAGATGTAACATTTGACCCAGCAAATACTATTGCAGACAGTGTAACAGCAATTTCA
GGATTAGTGAACGTGTATTACGATATAGCAATTGAAGATATGCCTATCACTCCATACATTGGTGTTGGTGT
TGGTGCAGCGTATATTAGCACTCCTTTGGAACCCGCTGTGAATGATCAAAAAAGTAAATTTGGTTTTGCTG
GTCAAGTAAAAGCTGGTGTTAGTTATGATGTAACTCCAGAAGTCAAACTTTATGCTGGAGCTCGTTATTTC
GGTTCTTATGGTGCTAATTTTGATGGAAAAAAAACAGATCCTAAAAATTCAACCGGACAGGCTGCTGATGC
AGGCGCATACAAAGTT

>HVR1
TKVDGITYKKDKSDYSPLKPSFIAGGGAFGYKM


>HVR2
DDIRVDVEGVYSYLNKNDVKDVTFDPANTIADSVTAISGLVNVYYDIAIE

>HVR3
DMPITPYIGVGVGAAYISTPLEPAVNDQKSKFGFAGQVKAGVSYDVTPEVK


>HVR4
LYAGARYFGSYGANFDGKKTDPKNSTGQAADAGAYKV

Note: the four HVRs peptides are consecutive in the protein sequence.

Protocol for typing and Submission of data

Typing a wsp sequence means to assign each unique HVR peptide a number and thus each wsp sequence a four number-combination (an HVR profile).

  1. Preparation of the data

    Prepare the following FASTA files with your sequences:

    • wsp nucleotide sequences
    • HVR1 amino acid
    • HVR2 amino acid
    • HVR3 amino acid
    • HVR4 amino acid

    To prepare the wsp nucleotide sequences, make a rough alignment of all your sequences using the alignment 'wsp.fas' as reference (from Baldo et al. 2005). Include the correct range of nucleotides indicated. Only align conserved regions.

    To prepare the four HVR sequence files, translate the wsp nucleotide alignment you just generated into proteins and then cut the sequences into four consecutive portions using the templates above as reference for the correct range of amino acids to include in each file.

  2. wsp allele and HVR peptide assignment

    This step is necessary for you to verify:

    for each of your wsp nucleotide sequences and HVR peptides whether

    1. It is an allele/ peptide already present in the database. In this case the sequence has been already assigned an allele/peptide number.
    2. It is a new allele/peptide. A sequence is considered a novel allele/peptide if is has at least one site difference compared to any other sequence already in the database. In such a case the new sequence has to be inserted into the database as a new allele/peptide and assigned a number.

    Do a batch query of wsp nucleotide sequences as follows:

    • Go to the Wolbachia home page (http://pubmlst.org/wolbachia/) and click on 'Sequence and profile definitions'.
    • Click on 'Batch sequence query'.
    • Select locus query with 'wsp'.
    • Copy all wsp nucleotide sequences from the wsp alignment you prepared and paste them into the text box.
    • Click Submit. The program will search the wsp nucleotide database for the best allele match to each of your query sequences.

    The Results table will indicate one of the following:

    • 'nucleotide allele' followed by a number. This is the case when a perfect match exists between your query sequence and an allele already into the database. For each strain that shows a perfect allele match, take note of the given allele number for the field 'wsp' in the Isolates form.
    • 'allele' followed by a number and a link to BLAST results. This is the case when a match exists between your query sequence and a matching sequence in the database, but only for a portion of your query sequence. This may occur if the query sequence does not contain the standard range of nucleotides required (check the templates as reference).
    • 'partial match' followed by the allele number and the percentage of identity of the closest match. Please note that 100% identity can be given for partial matches for reasons as above. All sequences that give partial matches are new alleles.
      • Prepare a FASTA file including all the new wsp nucleotide alleles. Eliminate all redundant sequences. To do that you can use a web tool in the MLST home page (Go to http://pubmlst.org/analysis/, then click on Non-redundant databases (NRDB)).

        Do a batch query of each HVR peptides sequences as follows:

      • Go to the Wolbachia wsp home page (http://pubmlst.org/wolbachia/) and click on 'Sequence and profile definitions'.
      • Click on 'Batch sequence query'.
      • Select locus query with 'hvr1'.
      • Copy all HVR1 amino acid sequences from the HVR1 alignment you prepared and paste them into the search box.
      • Click Submit. The program will search that hvr1 amino acid database for the best peptide match to each of your query sequences.

        The Results table will indicate one of the following:

        • 'peptide' followed by a number. This is the case when a perfect match exists between your query sequence and a peptide already into the database. For each strain take note of the given peptide number for the appropriate field in the Isolates form (i.e., 'HVR1').
        • 'peptide' followed by a number and a link to BLAST results. This is the case when 100% identity exists between your query sequence and a matching sequence in the database, but only for a portion of your query sequence. This may occur if the query sequence has not the standard range of nucleotides required (then check again the templates as reference).
        • 'partial match' followed by the peptide number and the percentage of identity of the closest match. Please note that 100% identity can be given for partial matches for reasons as above. All sequences that give partial matches are new peptides.
      • Query HVR2, 3 and 4 sequences as above.
      • Prepare a FASTA file including all the new HVR peptides. Eliminate all redundant sequences.
  3. Submission of data and completion of profiles

    Send the following files to the curator (amanda.r.avery@rochester.edu):

    • Forward and reverse trace files of the new wsp sequences
    • FASTA files containing:
      • new wsp sequences (in nucleotides)
      • new HVR1, HVR2, HVR3 and HVR4 peptides (in amino acids).

    The curator will check sequence quality and submit all new sequences to the database.

    After receiving a confirmation e-mail, re-query all your wsp nucleotide sequences as follows:

    • Go to the Wolbachia home page (http://pubmlst.org/wolbachia/) and click on 'Sequence and profile definitions'.
    • Click on 'Single sequence query'.
    • Select locus query with 'all'.
    • Copy and paste a single wsp nucleotide sequences into the search box.
    • Click Submit. The program will search the tables for the best nucleotide and HVR peptide match to your query sequence.

    Identical matches should be given for the wsp allele and each HVR. Complete filling of the fields 'wsp', 'hvr1', 'hvr2', 'hvr3', and 'hvr4' in the Isolates form.

Citing the database

The preferred format for citing this website in publications is:

This publication made use of the MLST website (https://pubmlst.org/ wolbachia/) sited at the University of Oxford (Jolley & Maiden 2010, BMC Bioinformatics, 11:593). The development and maintenance of this site has been funded by the Wellcome Trust.

Citation for use of the Wolbachia MLST system and database is Baldo et al. 2006, Appl Environ Microbiol 72:7098-7110. Development and curation of the Wolbachia MLST system has been supported by funds from the US National Science Foundation to Jack Werren as part of community outreach activities.

Status

Sequence database
Sequences: 3,536
Profiles (MLST): 524
Last updated: 2019-07-09

Isolate database
Isolates: 1,920
Last updated: 2019-04-19