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When selecting a variant of LR3, LR3-I covered 100% of 1000 randomly selected aligned fungal sequences from Genbank.

Comparison of the original LR3 primer to the LR3-I primer used for this experiment. The resulting LR3-I primer was tested in silico against the complete National Center for Biotechnology Information's (NCBI MD, USA) nucleotide database to confirm comparable coverage and specificity to the original LR3 primer. The conserved site that was selected for the LR3-I primer overlapped part of the original LR3 primer, but shifted four nucleotides over to the 5′-end and excluded the last three nucleotides on the 3′-end ( Figure 1).

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To design the LR3-I primer, a sample of 1000 fungal sequences from GenBank were aligned using CLC Genomic Workbench software (Qiagen, Hilden, Germany ) to identify a highly conserved region near the LR3 binding site. The variant LR3-I (5′ TGG TCC GTG TTT CAA GAC 3′) was created due to the high failure rate of the original LR3 primer during PCR amplification when the ONT barcoding tag was attached to the 5′-end. This primer pair targets the internal transcribed spacer (ITS) region and several hundred bases in the LSU region. The reverse primer was a variant of the LR3 primer (5′ CCG TGT TTC AAG ACG GG 3′), which binds on the 5′-end of the rRNA gene large subunit region (LSU) and also provides broad taxonomic coverage. The forward primer selected for this experiment was ITS1f-Kyo2 (5′ TAG AGG AAG TAA AAG TCG TAA 3′), based on its greater taxonomic coverage of fungi. Both primers were ordered with ONT barcoding tags, which are required for subsequent barcode attachment. Extracted gDNA was successfully amplified using the forward and reverse primers. All DNA extractions were performed using an optimized protocol (see Supplementary Methods 1) for the Synergy™ 2.0 Plant DNA Extraction kit (OPS Diagnostics, NJ, USA). These 16 members represented mycorrhiza, saprotrophs, pathogens and endophytes. To design the three mock libraries (Mock A, B and C), a total of 16 species from 12 genera representing 11 families from Basidiomycota, Ascomycota and Mortierellomycotina (see ) were collected or cultured from environmental samples (e.g., roots and leaves). Taxon affiliation, sample origin and material extracted from 16 taxa used to prepare mock communities. The aim of this experiment was 1) to develop an applicable pipeline to analyze MinION 1D sequence data and 2) to determine if 1D reads from the MinION could accurately identify a diversity of environmentally derived fungal taxa. The libraries were assembled from DNA extracted from sporocarps or cultures directly isolated from leaf and root tissue. The three libraries contained 16 taxonomically diverse species ( Table 1). To test the ability of the MinION in identifying fungi to the species level and distinguishing closely related species, three barcoded mock libraries were sequenced (Mock A, B and C), and processed through our selected pipeline.

Despite these advancements, there still remains a paucity of information regarding the capacity of the MinION for identifying a broad range of fungal diversity using amplicon sequencing. Although challenges still remain with DNA/RNA extraction, PCR and sequencing protocols in remote environments, the MinION has also been successfully executed in remote pop-up labs, ranging from Arctic Canada to the Amazon Rainforest . The MinION has also proved to be successful in genome assembly of fungal species such as Leptosphaeria, Sacchyaromyces and Candida spp. It has the ability to generate longer reads and is efficient in determining bacterial microbiomes. The Oxford Nanopore Technologies (ONT) MinION™ sequencer is a small third-generation HTS platform that became commercially available in 2015. For example, they provide longer sequence reads, and despite the higher error rates (5–13%, ), high-quality reference data can be generated and species-level identification can be inferred by averaging out the errors with a consensus from multiple reads. However, the ongoing improvements of third-generation HTSs hold promise for diversity analyses of environmental samples.

Despite these capabilities, established HTS methods (e.g., Illumina and Pyrosequencing) are limited in taxonomic resolution because of their shorter sequence reads and the need for expensive analytical technology.

High-throughput sequencing (HTS) provides the ability to multiplex and profile fungal communities across environmental samples, including but not limited to leaf tissue, soil and roots.

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