Work

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

Year: 2026

Type: article

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.

Source: Frontiers in Microbiology

DOI: 10.3389/fmicb.2026.1904425