Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing.
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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.
Next generation sequencing (NGS)
PacBio CCS sequencing
High-Throughput Nucleotide Sequencing
Multilocus Sequence Typing
Polymerase Chain Reaction
Published Version (Please cite this version)10.1016/j.fgb.2015.01.005
Publication InfoChen, Yuan; Frazzitta, Aubrey E; Litvintseva, Anastasia P; Fang, Charles; Mitchell, Thomas G; Springer, Deborah J; ... Perfect, John R (2015). Next generation multilocus sequence typing (NGMLST) and the analytical software program MLSTEZ enable efficient, cost-effective, high-throughput, multilocus sequencing typing. Fungal Genet Biol, 75. pp. 64-71. 10.1016/j.fgb.2015.01.005. Retrieved from https://hdl.handle.net/10161/11056.
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Associate Professor Emeritus in Molecular Genetics and Microbiology
Among patients with AIDS, leukemia or other cancers, organ or bone marrow transplants, and similar immunocompromising risk factors, the incidence of opportunistic mycoses and the number of different fungal pathogens are increasing dramatically. For many of these fungi, the definition of a species and the recognition of pathogen are highly problematic. Conventional methods of identification are based on morphological and physiological characteristics and are often time-consuming, difficult
James B. Duke Distinguished Professor of Medicine
Research in my laboratory focuses around several aspects of medical mycology. We are investigating antifungal agents (new and old) in animal models of candida and cryptococcal infections. We have examined clinical correlation of in vitro antifungal susceptibility testing and with in vivo outcome. Our basic science project examines the molecular pathogenesis of cryptococcal infections. We have developed a molecular foundation for C. neoformans, including transformation systems, gene disr
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