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

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.

Keywords: DNA sequencing; diagnostic; fungal.

FIG 1

Phylogenetic relationships of strains belonging to the Cryptococcus gattii and C. neoformans species complexes inferred from maximum-likelihood analysis of IGS sequences. Bayesian posterior probabilities (PP) (left) and maximum-likelihood bootstrap support (BT) (right) are shown on the nodes. The tree is rooted with the corresponding IGS sequence from Cryptococcus wingfieldii strain CBS 7118. Asterisks represent reference strains.

FIG 2

Phylogenetic relationships of species belonging to the C. gattii and C. neoformans species complexes inferred from maximum-likelihood analysis of IGS sequences using the nanopore (R10.3 flow cell) and Sanger methods. Bayesian posterior probabilities (PP) (left) and maximum-likelihood bootstrap support (BT) (right) are shown on the nodes. The tree is rooted with Cryptococcus wingfieldii CBS 7118. Asterisks represent reference strains.