title: Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing.
abstract: Multilocus sequence typing (MLST) has become the preferred method for genotyping many biological species, and it is especially useful for analyzing haploid eukaryotes. MLST is rigorous, reproducible, and informative, and MLST genotyping has been shown to identify major phylogenetic clades, molecular groups, or subpopulations of a species, as well as individual strains or clones. MLST molecular types often correlate with important phenotypes. Conventional MLST involves the extraction of genomic DNA and the amplification by PCR of several conserved, unlinked gene sequences from a sample of isolates of the taxon under investigation. In some cases, as few as three loci are sufficient to yield definitive results. The amplicons are sequenced, aligned, and compared by phylogenetic methods to distinguish statistically significant differences among individuals and clades. Although MLST is simpler, faster, and less expensive than whole genome sequencing, it is more costly and time-consuming than less reliable genotyping methods (e.g. amplified fragment length polymorphisms). Here, we describe a new MLST method that uses next-generation sequencing, a multiplexing protocol, and appropriate analytical software to provide accurate, rapid, and economical MLST genotyping of 96 or more isolates in single assay. We demonstrate this methodology by genotyping isolates of the well-characterized, human pathogenic yeast Cryptococcus neoformans.
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, NC, USA; Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA. Electronic address: ychenbioinfo@gmail.com.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, NC, USA.
authors.forename: Aubrey E
authors.initials: AE
authors.lastname: Frazzitta
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Anastasia P
authors.initials: AP
authors.lastname: Litvintseva
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, NC, USA.
authors.forename: Charles
authors.initials: C
authors.lastname: Fang
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Thomas G
authors.initials: TG
authors.lastname: Mitchell
authors.affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC, USA.
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Springer
authors.affiliation: Janelia Research Campus, HHMI, Ashburn, VA, USA.
authors.forename: Yun
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Pacific Sciences, Menlo Park, CA, USA.
authors.forename: George
authors.initials: G
authors.lastname: Yuan
authors.affiliation: Division of Infectious Diseases, Department of Medicine, Duke University Medical Center, Durham, NC, USA. Electronic address: perfe001@mc.duke.edu.
authors.forename: John R
authors.initials: JR
authors.lastname: Perfect
mesh_terms: D003362:Cost-Benefit Analysis; D003455:Cryptococcus neoformans / Q000145:classification* / Q000235:genetics*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods*; D006801:Humans; D058885:Multilocus Sequence Typing / Q000191:economics / Q000379:methods*; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012984:Software
keywords: Cryptococcus neoformans; MLST; Multiplex PCR; Next generation sequencing (NGS); PacBio CCS sequencing; Software