Different amplicon targets for sequencing-based studies of fungal diversity

Target-gene amplicon sequencing is the most exploited high-throughput sequencing application in microbial ecology. The targets are taxonomically relevant genes with the 16S rRNA being the gold standard for bacteria. As for Fungi, the most commonly used target is the internal transcribed spacer (ITS). However, the uneven ITS length among species may promote preferential amplification and sequencing and incorrect estimation of their abundance. Therefore, the use of different targets is desirable. We evaluated the use of three different target amplicons for the characterization of fungal diversity. After an in silico primer evaluation, we compared three amplicons (ITS 1-2, 18S ribosomal small subunit RNA and D1/D2 domain of the 26S ribosomal large subunit RNA) both using biological samples and a mock community of common fungal species. All the three targets allowed an accurate identification of the species present. Nevertheless, high heterogeneity in ITS 1-2 length was found and this caused an over-estimation of the abundance of species with shorter ITS, while both 18S and 26S amplicons allowed a more reliable quantification. We demonstrated that ITS 1-2 amplicon sequencing, although widely used, may lead to an incorrect evaluation of fungal communities and efforts should be done for promoting the use of different targets in sequencing-based microbial ecology studies.Importance Amplicon-sequencing approaches for fungi may rely on different targets affecting diversity and abundance of the fungal species. An increasing number of studies will address the fungal diversity by high-throughput amplicon sequencing. The description of the communities must be accurate and reliable in order to draw useful insights and to address both ecological and biological questions. By analysing a mock community and several biological samples we demonstrate that using different amplicon targets may change the results of fungal microbiota composition and we highlight how a careful choice of the target is fundamental for a thorough description of the fungal communities.