In addition, Nilsson et al.  showed that about 20% of the fungal DNA sequences from the public sequence databases may be identified to incorrect species, and that the majority of entries lack descriptive and up-to-date annotations. However, our analyses deal with taxonomic groups at the sub-kingdom/phylum level (basidiomycetes,
ascomycetes and ‘non-dikarya fungi’) and it is unlikely that those classes suffer significantly from incorrect identifications (e.g. that ascomycetes have been accessioned as basidiomycetes). The fact that no ascomycete sequences were amplified using primer ITS4-B, even when allowing 3 mismatches (Table 1), also supports the reliability of the conclusions in this respect. All the investigated primers were hampered by some mismatches relative to the target sequences in AZD1152 in vitro Stem Cells inhibitor subsets 1-3, and they also varied in their specificity to fungi versus plants. It is noteworthy that ITS1-F, which is frequently used in fungal environmental sequencing studies and assumed to be fungal specific , only amplified three plant sequences after removing the fungal sequences erroneously deposited as plants. Those three sequences deposited as plants most probably corresponded to errors as well. However, the ITS1-F primer is hampered with a high degree of mismatches.
Our analysis indicates that it may be important to use this primer under relaxed PCR conditions when targeting all fungi in an environmental sample. We confirmed that the primer ITS4-B, which has also often been used
in environmental sequencing studies (e.g. [8, 28, 30, 31]), is very specific to basidiomycetes, as it did not amplify plant ITS even under relaxed PCR conditions. However, this primer is only able to target a small proportion of the basidiomycete diversity (Table 4). Mainly Boletales and a fraction of the Agaricales are amplified Teicoplanin under strict conditions, while under relaxed conditions, Chantharellales, Hymenochaetales, Tremellomycetes, Polyporales and Russulales are amplified to a certain degree (from 28 to 94% depending on the group). Pucciniomycotina and Ustilaginomycotina are not amplified at all. Hence, our in silico analyses indicate that ITS4-B should be used with great caution or perhaps abandoned completely in environmental sequencing studies where the aim is to characterize the diversity of all basidiomycetes. Although not specific to fungi, the primer pairs ITS5-ITS2 and ITS3-ITS4 apparently have a better ability to amplify fungal ITS as the proportion of sequences amplified does not vary much between strict and relaxed PCR conditions. Overall, the results indicate that it is important to assess the specificity of the amplification in relation to PCR stringency before interpreting the results from environmental samples in terms of abundance and diversity.