Fungal Mock Community with ITS amplicon

The Oxford Nanopore Technologies’ ITS amplicon sequencing method can be used to profile complex fungal communities. This post describes a new fungal dataset release, using newly released ITS Primers included in the Microbial Amplicon Barcoding Kit 24 V14 (SQK-MAB114.24). The dataset was generated from the ATCC Mycobiome Genomic DNA Mix following the Microbial Amplicon Barcoding protocol, and sequenced on MinION Flow Cells on a GridION device. ​

The fungal mock community comprises an even mix of ten fungal strains, including: Aspergillus fumigatus, Cryptococcus neoformans, Trichophyton interdigitale, Penicillium chrysogenum, Fusarium keratoplasticum, Candida albicans, Nakaseomyces glabratus, Malassezia globosa, Saccharomyces cerevisiae and Cutaneotrichosporon dermatis. This dataset provides a valuable resource for evaluating fungal community analysis methods. This is illustrated in the supplied analysis example, where the data are processed with the EPI2ME wf-16s workflow.

Sample

Preparation

Sample preparation was performed according to the Microbial Amplicon Barcoding Kit 24 V14 Protocol published on the Oxford Nanopore Technologies website.

Further preparation information such as sample storage suggestions can be found on the Oxford Nanopore Website.

Sequencing

Sequence data was generated using the following configuration:

Data Download

The dataset is available for anonymous download, without login, from a public Amazon Web Services S3 bucket. The bucket is part of the Open Data on AWS project enabling sharing and analysis of a wide range of data. The data can be downloaded with the AWS CLI command:

aws s3 sync --no-sign-request s3://ont-open-data/fungal_ITS_2025.09 fungal_ITS_2025.09

See the tutorials page for information on downloading the dataset.

You can also browse and download the files in your web browser courtesy of 42basepairs.

Analysis

Analysis outputs are available. The analysis results are located in the S3 bucket under the prefix:

s3://ont-open-data/fungal_ITS_2025.09/analysis

EPI2ME workflows used to generate analysis outputs:

• wf-16s (v1.6.0)

Related Materials

• Microbial Amplicon Barcoding Kit 24 V14 Protocol
• Purchase the Microbial Amplicon Barcoding Kit 24 V14
• Knowledge exchange webinar: How to sequence 16s and ITS microbial amplicons