Lott, M. E. J. | bioRxiv Science

bioRxiv2026

Benchmarking Short-Read ITS2 and Full-Length ITS Sequencing Reveals Pipeline-Dependent Biases in Indoor Fungal Community Profiling

Short-read amplicon sequencing is widely used for fungal surveys but can limit taxonomic resolution. Long-read sequencing enables recovery of the full internal transcribed spacer (ITS) region and may improve ecological and taxonomic inference. Here, we conducted a paired comparison of Illumina ITS2 and PacBio HiFi full-length ITS sequencing using identical DNA extracts from built-environmental air and surface samples (n = 68) collected across homes, a dormitory, and laboratories. Both datasets were taxonomically assigned using the same algorithm and reference database. We performed paired statistics, in-silico ITS2 trimming of long-read sequences, and cross-platform mapping at multiple identity thresholds. Full-length ITS provided higher taxonomic resolution, assigning a greater fraction of ASVs at the family (98% vs. 88%) and species (42% vs. 32%) ranks than ITS2 (paired Wilcoxon q = 0.002). Alpha-diversity comparisons showed similar Shannon diversity across pipelines, whereas richness metrics were consistently higher for full-length ITS. Beta-diversity analyses indicated broadly comparable community-level patterns, although full-length ITS revealed stronger sample-type- and location-associated structure (PERMANOVA R{superscript 2} [≥] 0.06, p = 0.0001). In-silico ITS2 trimming reduced these differences, indicating that amplicon length is a major contributor to enhanced taxonomic resolution and ecological inference. Cross-platform mapping further showed extensive one-to-many relationships between ITS2 and full-length ITS ASVs, consistent with increased sequence resolution in long-read data. Together, these results show that ITS2 sequencing provides robust community-level profiling, while full-length ITS enables improved richness estimates and finer ecological and taxonomic resolution. This paired, bias-aware framework provides a practical template for selecting fungal amplicon sequencing strategies in built-environment mycobiome studies. ImportanceFungal communities in built environments influence indoor air quality and human exposure, yet their characterization depends strongly on sequencing strategy. This study provides a controlled, paired comparison of short-read ITS2 and long-read full-length ITS sequencing, showing that differences in amplicon length substantially contribute to variation in taxonomic resolution and ecological inference. While both approaches yield comparable community-level patterns, full-length ITS improves richness estimates, species-level assignment, and environmental discrimination by resolving sequence variation collapsed in ITS2 surveys. By integrating paired diversity analyses, in-silico ITS2 trimming, and cross-platform ASV mapping, this work offers a bias-aware framework for evaluating fungal amplicon pipelines. Importantly, improved species-level resolution enables functional interpretation of indoor fungi, for example the identification of taxa associated with pathogenic traits, allergen production, or toxin synthesis, supporting the development of more informative exposure metrics and targeted assays relevant to human health in built environments.

microbiology↗

bioRxiv2025

Comparative Genomics of Vibrio vulnificus, Vibrio navarrensis, and Vibrio cidicii Reveals Taxonomic Boundaries and Divergent Virulence Mechanisms

Vibrionaceae are a diverse family of bacteria that contain pathogenic species, including those within the Vulnificus clade: Vibrio vulnificus, Vibrio navarrensis, and Vibrio cidicii. While V. vulnificus is a generally well characterized environmental pathogen, V. cidicii and V. navarrensis are relatively rare, recently identified species that our current understanding of virulence and environmental adaptation is limited. Here, we investigate genetic relatedness across these three species to identify shared and species-specific genes, including markers of virulence using publicly available genome assemblies. We evaluated phylogenetic and genomic diversity across this clade by sampling all available V. navarrensis and V. cidicii genomes, and a biodiverse curated set of four V. vulnificus ecotypes to ensure representative coverage. Our results indicate that all three species share 2,321 universally conserved genes, many of which are core bacterial functions. Moreover, V. cidicii and V. navarrensis have extensive genetic similarity between them, including average nucleotide identities >95% and 619 shared genes. Despite this similarity, they both remain more phylogenetically distant from V. vulnificus and lack key virulence genes such as rtxA, indicating alternative pathogenic mechanisms. Overall, these findings reveal that virulence potential varies across the clade and environmental adaptation potential varies between species and biotypes. IMPORTANCEVibrio species are important environmental aquatic bacteria that pose a threat to human and animal health across the globe. This study applied comparative genomics to investigate the genetic relatedness of Vibrio vulnificus, Vibrio navarrensis, and Vibrio cidicii, with special focus on genes associated with environmental adaptation and virulence between and within each species. Results indicate V. navarrensis and V. cidicii share many genes and are phylogenetically close, and that they possess different virulence potential than V. vulnificus. This adds to our understanding of genetic diversity and pathogenic mechanisms within an important group of marine pathogens.

microbiology↗