Methodology comparison of environmental sediment fungal community analysis

Abstract

Fungi play important roles in ecosystems. Analyzing fungal communities in environments has long been a challenge due to the large difference in compositions retrieved using different methods or sequencing regions, obscuring the true abundance and species information. Our study aimed to compare and determine more accurate approach for evaluating fungal populations in river sediment. To achieve this, different primer sets in the internal transcribed spacer (ITS) (ITS5/ITS1R, ITS1F/ITS2), 18S rRNA gene (0817F/1196R) for High-throughput sequencing (HTS), metagenomic shotgun sequencing (MS) directly from environmental samples, and HTS using ITS primers for the fungal samples collected from plate cultivation were used to characterize the fungal communities. We calculated diversity index and used FungalTraits to analyze methods preferences for fungal species. The study revealed that when analyzing the fungal species directly from environmental samples, amplification and sequencing of ITS region demonstrated more accuracy than MS and 18S rRNA gene sequencing methods, but displayed significant primer preference. Over 30 % fungal species from HTS after plate cultivation were not present in HTS from the environmental samples. NMDS analysis demonstrated significant disparities in species diversity among different methods, suggesting potential complementarity between them. Over 85% species identified by HTS using ITS primers belonged to filamentous fungi, while the MS mostly identified yeast (62%). Therefore, to get more accurate fungal community information in sediment, multiple methods were recommended by using cultivation, molecular biological methods dependent on PCR techniques like ITS1F/ITS2 primer for HTS and PCR independent method such as metagenomic shotgun sequencing techniques.