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.
Type
Journal articleSubject
Cryptococcus neoformansMLST
Multiplex PCR
Next generation sequencing (NGS)
PacBio CCS sequencing
Software
Cost-Benefit Analysis
Cryptococcus neoformans
Genotype
High-Throughput Nucleotide Sequencing
Humans
Multilocus Sequence Typing
Phylogeny
Polymerase Chain Reaction
Software
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https://hdl.handle.net/10161/11056Published Version (Please cite this version)
10.1016/j.fgb.2015.01.005Publication Info
Chen, 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.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Thomas Greenfield Mitchell
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
John Robert Perfect
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
Alphabetical list of authors with Scholars@Duke profiles.

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Rights for Collection: Scholarly Articles
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