Targeted long-read eukaryotic profiling of human stool by broad-range 18S–28S rRNA gene amplicon sequencing on Oxford Nanopore Technologies: a proof-of-concept study
Abstract
The gut eukaryome, comprising the protists and fungi that fall within the strict definition of the microbiota together with the macroscopic helminths and arthropods that lie outside it, is under-detected by clinical microscopy and by short-read 16S/18S sequencing, yet is increasingly implicated in inflammatory and autoimmune disorders, including psoriasis. Existing 18S V4/V9 primers either lack cross-kingdom universality or fail to discriminate closely related species. We designed and evaluated broad-range PCR primers anchored in conserved blocks of nuclear 18S and 28S ribosomal genes, flanking a 1.0–3.5 kb cassette spanning the 3′-end of 18S, ITS1, 5.8S, ITS2, and the 5′-end of 28S. Stool samples from 91 plaque-psoriasis patients and 92 matched controls were collected; a subset of 32 samples was profiled by Oxford Nanopore amplicon sequencing using the EPI2ME wf-metagenomics pipeline (Kraken + Bracken) against the NCBI core_nt database. The gut eukaryome was dominated by Ascomycota and Protista, with 51 species-level entries assigned to Candida (37 named binomials), 27 entries assigned to Blastocystis (12 subtype-labelled entries), 20 entries assigned to Malassezia (15 named species), and 37 entries assigned to Cladosporium recovered from a single PCR tube. Blastocystis hominis was detected in 37.5% of subjects (≥50-read threshold; mean 7.5% in carriers), with concurrent detection of 12 subtype-labelled entries (ST1-ST4, ST7, ST10-ST15, ST24) and 11 named isolate entries. Psoriasis-relevant Candida species, such as C. albicans (34.4%), C. tropicalis (34.4%), C. parapsilosis (31.2%), and Malassezia restricta (9.4%; up to 12.1%) were co-detected. Long-read sequencing of a broad-range 18S -28S rRNA gene cassette provides a single-tube workflow for the simultaneous recovery of fungal, protistan, and plant-derived eukaryotic sequences from human stool.