Benchmarking full-length ITS metabarcoding across Illumina 2x500, PacBio Revio, and Oxford Nanopore sequencing using mock and soil communities
High-throughput sequencing of the full-length internal transcribed spacer (ITS) marker can improve taxonomic resolution for fungi and other eukaryotes, but sequencing platform choice and bioinformatics can strongly affect inferred diversity. We generated full-length ITS amplicon datasets from a multi-species fungal mock community comprising pooled DNA from 103 fruiting body specimens and 45 composite soil samples spanning cropland, grassland and forest sites, with three field sampling designs per site. Amplicons were sequenced using Illumina MiSeq i100 2x500 paired-end sequencing, PacBio Revio HiFi (CCS) sequencing and Oxford Nanopore MinION long-read sequencing, including technical PCR variants (30 and 35 cycles).
High-throughput sequencing of the full-length internal transcribed spacer (ITS) marker can improve taxonomic resolution for fungi and other eukaryotes, but sequencing platform choice and bioinformatics can strongly affect inferred diversity. We generated full-length ITS amplicon datasets from a multi-species fungal mock community comprising pooled DNA from 103 fruiting body specimens and 45 composite soil samples spanning cropland, grassland and forest sites, with three field sampling designs per site. Amplicons were sequenced using Illumina MiSeq i100 2x500 paired-end sequencing, PacBio Revio HiFi (CCS) sequencing and Oxford Nanopore MinION long-read sequencing, including technical PCR variants (30 and 35 cycles).
University of Tartu