Multilocus sequence typing (MLST).
Multilocus sequence typing is a technique for subtyping strains that is applied mainly to microorganisms. It consists of a "wet" stage (PCR amplification and sequencing of 7-10 housekeeping genes) followed by a "dry" stage (assembly of sequence traces to alleles, online allele assignment, and designation of strain sequence types). The MLST facilities are set up for processing strains in 96-well microtitre plates from strain DNA extracts through to finished sequencing reactions, then from DNA sequence output files through to strain sequence types. Wet-stage facilities comprise a Beckman Biomek 2000 liquid handling robot, a microtitre plate sealer, 8 PCR blocks, agarose electrophoresis for the analysis of 96 PCR products, and centrifuges and automated multichannel pipettes for post-PCR cleanup. To minimise PCR carryover contamination, the wet stage is segregated into pre-PCR (arraying of strain DNA, setup of PCR and sequencing reactions) and post-PCR (gel monitoring of PCR products, cleanup of PCR and sequencing reactions) stages carried out in different rooms. The dry stage only requires access to a networked PC and the installation of specific freeware (currently STARS, though upgrade to PHINEUS is expected before the end of 2007. MLST of one 96-well plate of isolates for 8 genes from DNA extracts in single tubes through to designated strain sequence types takes one full-time researcher 2.5 days for the wet stage and 0.5 days for the dry stage.