Multilocus Sequence Typing Protocol for Cryptococcus neoformans

Seven MLST loci (CAP59, GPD1, IGS1, LAC1, PLB1, SOD1, and URA5) were amplified and sequenced following the procedures of the International Society for Human and Animal Mycology (ISHAM) MLST consensus typing scheme for C. neoformans (http://mlst.mycologylab.org).^{25 (link)} Sequencing was performed using BigDye v3.1 Chemistry (Applied Biosystems, CA, USA) on an ABI 3130 Genetic Analyzer (Applied Biosystems, CA, USA). Both forward and reverse amplicons of each locus were sequenced. Consensus sequences were manually edited using ContigExpress and aligned in AlignX, implemented in VectorNTI Suite 7.0.^{26 (link)} A single Allele Type (AT) number was assigned to each of the seven loci by comparing consensus DNA sequences with the ISHAM database, resulting in a seven-number allelic profile for each isolate. The allelic profiles defined the corresponding STs.
MLST profiles and DNA sequences at each MLST loci for isolates from regions other than Laos and Vietnam were obtained from NCBI. For the global analysis, we used data reported by Simwami et al.,^{21 (link)} Mihara et al.,^{14 (link)} and Cogliati et al.^{27 (link)} included in Khayhan et al.,^{17 (link)} and from Chen et al.,^{28 (link)} Ferreira-Paim et al.,^{29 (link)} Beale et al.,^{30 (link)} and Dou et al.^{31 (link)}