MLST 1.8 Instructions

To execute a sequence type prediction using the MLST web-server you need to follow the following 4 steps:

1. Choose the MLST profile for your organism

Use the drop down list to select the MLST scheme. For most organisms only one MLST scheme is available.
For four organisms, two different MLST schemes are available:

• Acinetobacter baumannii (Acinetobacter baumannii#1 [1], Acinetobacter baumannii#2 (link)).
• Clostridium difficile (Clostridium difficile#1 [2], Clostridium difficile#2 [3]).
• Escherichia coli (Escherichia coli#1 [4], Escherichia coli#2 [5]).
• Pasteurella multocida (Pasteurella multocida (RIRDC), Pasteurella multocida#2 (multihost)).

2. Select sequencing technology (or choose assembled genome)

See section 3 for more details on supported technologies and supported file formats.

3. Upload the file(s) of the isolate, or annotated genome, for which you want to know the sequence type.

The file to upload must contain assembled contigs, raw reads from the chosen sequencing technology or an annotated genome (among those shown in the mlst scheme drop list).

For preassembled partial or complete genomes, the input sequence must be in one-letter nucleotide code in a single FASTA file. The allowed alphabet (not case sensitive) is the following:

A C G T and N (unknown)

To input the sequences, upload a single FASTA file on your local disk by using the applet. First click on Isolate file then choose your file and click Open as shown in the image at the bottom of the page.

If your input consists of unassembled short sequence reads, the MLST web-server will support the following technologies:

• Illumina Solexa single end reads
• Illumina Solexa paired end reads
• Roche 454 single end reads
• Roche 454 paired end reads
• Ion-torrent single end reads
• SOLiD single end reads
• SOLiD paired end reads
• SOLiD mate paired reads

Below, the number and format of the files that must be uploaded is specified. If two files are to be uploaded for paired end reads you can select both files with Ctrl-Click (or Cmd-Click on Mac).
Note: From here on, se denotes single end reads, pe denotes paired end reads, and mp denotes mate paired reads.

1.1 - Illumina Solexa

The assembly of raw reads obtained by Illumina Solexa machines is supported, and in particular, single end and paired end reads are supported.
Supported file formats: FASTA, FASTAQ
option: Illumina se (single end read)
In this case you will need to upload only one file in the supported format.
option: Illumina pe (paired end reads)
Two files of the supported formats must be uploaded.

1.2 - Roche GS (454)

The assembly of raw reads obtained by Roche GS (454) machines are supported, and in particular single end and paired end reads are supported.
Supported file formats: FASTA, FASTQ, fna, sff
option: 454 se (single end read)
In this case either a FASTA, FASTQ or a 454 sff raw file must be uploaded.
option: 454 pe (paired end reads)
In this case one or more FASTA, FASTQ and(or) 454 sff raw files must be uploaded.
IMPORTANT: In the 454 pe assembly the user must respect the supported file formats and extensions. this means that, supposing that my reads are called read1 and read2, and they 454 sff raw reads, than the file name must contain the sff extension, so the files to upload will be named read1.sff and read2.sff respectively.

1.3 - Ion-Torrent

The assembly of raw reads obtained by Life Science Ion Torrent machines is supported.
Supported file formats: FASTA, FASTQ
option: Ion Torrent se (single end reads)
The user can upload one or more files of the supported formats.

1.4 - Applied Biosystems SOLiD

The assembly of raw reads obtained by Applied Biosystems SOLiD sequencing machines is supported, and in particular single end, paired end and mate paired reads are supported.
Supported file formats: qual, cfasta
option: SOLiD se (single end read)
In this case a quality file and a raw read file (both of type R3) must be uploaded.
option: SOLiD pe (paired end reads)
2 quality files (one F3 and one R3) and 2 reads (one F3 type and one R3) must be uploaded to execute the assembly.
option: SOLiD mp (mate paired reads)
2 quality files (one F3 and one R5) and 2 reads (one F3 type and one R5) must be uploaded to execute the assembly.
IMPORTANT: The web sever will analyse and identify whether the uploaded files are quality files or colour-space raw reads. In this way the user does not need to worry about the file extention but still the files must be of the supported formats. Also, modification of the sequence headers in the files could cause the web-server not to function properly.

4. Submit the job

Click on the "Upload" button. The status of your job (either queued or running) will be displayed and constantly updated until it terminates and the server output page appears in your browser. You also have the option to input your email and be notified as soon as your the results are ready. The e-mail message will contain the URL under which the results are stored; they will remain on the server for 48 hours for you to collect them

An overview of the upload

REFERENCES

1. Bartual SG, Seifert H, Hippler C, Luzon MA, Wisplinghoff H, Rodriguez-Valera F. Development of a multilocus sequence typing scheme for characterization of clinical isolates of Acinetobacter baumannii. J Clin Microbiol 2005; 43:4382-90.
2. Griffiths D, Fawley W, Kachrimanidou M, et al. Multilocus sequence typing of Clostridium difficile. J Clin Microbiol 2010; 48:770-8.
3. Lemee L, Dhalluin A, Pestel-Caron M, Lemeland JF, Pons JL. Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types. J Clin Microbiol 2004; 42:2609-17.
4. Wirth T, Falush D, Lan R, et al. Sex and virulence in Escherichia coli: an evolutionary perspective. Mol Microbiol 2006; 60:1136-51.
5. Jaureguy F, Landraud L, Passet V, et al. Phylogenetic and genomic diversity of human bacteremic Escherichia coli strains. BMC Genomics 2008; 9:560.