Each gene is sequenced from individual strains and then compared against existing sequences in a publically accessible, globally maintained database. Those submitted sequences matching

ones already in the database are assigned the gene type number of the sequence in the database; if a novel sequence is submitted, the curator of the database assesses the sequencing results and assigns an appropriate gene number. While this approach does address several of the limitations encountered by other typing methods, the cost of sequencing #PF-04929113 solubility dmso randurls[1|1|,|CHEM1|]# can be a barrier to large scale typing projects. Particularly, because of the potential for error in sequencing reads the standard for determining a gene type requires matching forward and reverse sequences. The S. pneumoniae typing system is based on the partial sequence of seven genes coding for the housekeeping proteins: Shikimate dehyrogenase (aroE), glucose-6-phosphate dehydrogenase (gdh), glucose kinase (gki), transketolase (recP), signal peptidase I (spi), xanthine phosphoribosyltransferase

(xpt), and D-alanine-D-alanine ligase (ddl) [11]. Some preliminary results, and information provided by the Forskolin mouse curator of the S. pneumoniae MK-1775 nmr MLST database indicated that several of the provided MLST sequencing primers were unable to obtain the full sequence required in each direction. As a result, in cases where a novel gene type is identified based on sequences from the standard primers (Table 1), the investigators

are required to design new primers and re-sequence the particular gene (Cynthia Bishop, personal communication, May, 2012). In these circumstances, investigators are required to expend additional time and resources developing new primers, as well as purchasing additional sequencing and validating results. While several investigators in the field are aware of this issue, and all sequences in the MLST database have been correctly verified through subsequent primer redesign and re-sequencing, this limitation has not been specifically addressed in the literature [12, 13] (Cynthia Bishop, personal communication, May 2012). Table 1 Standard S.