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Mycology

Nanopore Sequencing of the Fungal Intergenic Spacer Sequence as a Potential Rapid Diagnostic Assay

Gretchen A. Morrison, Jianmin Fu, Grace C. Lee, Nathan P. Wiederhold, Connie F. Cañete-Gibas, Evelien M. Bunnik, Brian L. Wickes
Kimberly E. Hanson, Editor
Gretchen A. Morrison
aDepartment of Microbiology, Immunology, and Molecular Genetics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Jianmin Fu
aDepartment of Microbiology, Immunology, and Molecular Genetics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Grace C. Lee
bThe University of Texas at Austin, College of Pharmacy, Austin, Texas, USA
cPharmacotherapy Education and Research Center, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Nathan P. Wiederhold
dUTHSCSA Fungus Testing Laboratory, Departments of Pathology and Laboratory Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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  • ORCID record for Nathan P. Wiederhold
Connie F. Cañete-Gibas
dUTHSCSA Fungus Testing Laboratory, Departments of Pathology and Laboratory Medicine, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Evelien M. Bunnik
aDepartment of Microbiology, Immunology, and Molecular Genetics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Brian L. Wickes
aDepartment of Microbiology, Immunology, and Molecular Genetics, The University of Texas Health Science Center at San Antonio, San Antonio, Texas, USA
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Kimberly E. Hanson
University of Utah
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DOI: 10.1128/JCM.01972-20
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ABSTRACT

Fungal infections are being caused by a broadening spectrum of fungi, yet in many cases, identification to the species level is required for proper antifungal selection. We investigated the fungal intergenic spacer (IGS) sequence in combination with nanopore sequencing for fungal identification. We sequenced isolates from two Cryptococcus species complexes, C. gattii and C. neoformans, which are the main pathogenic members of this genus, using the Oxford Nanopore Technologies MinION device and Sanger sequencing. There is enough variation within the two complexes to argue for further resolution into separate species, which we wanted to see if nanopore sequencing could detect. Using the R9.4.1 flow cell, IGS sequence identities averaged 99.57% compared to Sanger sequences of the same region. When the newer R10.3 flow cell was used, accuracy increased to 99.83% identity compared to the same Sanger sequences. Nanopore sequencing errors were predominantly in regions of homopolymers, with G homopolymers displaying the largest number of errors and C homopolymers displaying the least. Phylogenetic analysis of the nanopore- and Sanger-derived sequences resulted in indistinguishable trees. Comparison of average percent identities between the C. gattii and C. neoformans species complexes resulted in only a 74 to 77% identity between the two complexes. Sequencing using the nanopore platform could be completed in less than an hour, and samples could be multiplexed in groups as large as 24 sequences in a single run. These results suggest that sequencing the IGS region using nanopore sequencing could be a potential new molecular diagnostic strategy.

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Nanopore Sequencing of the Fungal Intergenic Spacer Sequence as a Potential Rapid Diagnostic Assay
Gretchen A. Morrison, Jianmin Fu, Grace C. Lee, Nathan P. Wiederhold, Connie F. Cañete-Gibas, Evelien M. Bunnik, Brian L. Wickes
Journal of Clinical Microbiology Nov 2020, 58 (12) e01972-20; DOI: 10.1128/JCM.01972-20

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Nanopore Sequencing of the Fungal Intergenic Spacer Sequence as a Potential Rapid Diagnostic Assay
Gretchen A. Morrison, Jianmin Fu, Grace C. Lee, Nathan P. Wiederhold, Connie F. Cañete-Gibas, Evelien M. Bunnik, Brian L. Wickes
Journal of Clinical Microbiology Nov 2020, 58 (12) e01972-20; DOI: 10.1128/JCM.01972-20
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KEYWORDS

DNA sequencing
diagnostic
fungal

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