## 2 Materials and Methods
### Primers, PCRs, and Gel Electrophoresis  
Primers were designed according to recommendations for fungal metabarcoding [19] that, together with sample-specific index primers (with added adapters and overhangs as suggested by SciLifeLab National Genomics Infrastructure, NGI), enabled us to complete the design of extended primers for our sample library (Figure 1b). In total, we used five fungal-specific primers, four extended primers, and two primers for attachment to sample-specific index primers (Figure 1b). In general, 25\(\upmu\)L PCR mixture was used as consisting of 1X Dream Taq buffer, 0.16 \(\upmu\)M dNTP mix, 0.4 \(\upmu\)M forward and reverse primers, \(\sim\)0.25 \(\upmu\)g template DNA and 0.75 unit Dream Taq DNA polymerase and PCR grade water. Each PCR run was initiated with the following settings: initial denaturation at 95 \({}^{\circ}\)C for 3 min followed by 30 cycles of 95 \({}^{\circ}\)C for \begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Kit** & **Sample ID** & \multicolumn{2}{c}{**DNA Concentration**} & \multirow{2}{*}{**260/280**} & \multirow{2}{*}{**260/230**} \\  & & **(ng/\(\upmu\)L)** & & \\ \hline E.Z.N.A. Plant & 253 & 561.1 & 2.04 & 1.99 \\ DNA & 281 & 804.3 & 2.02 & 2.00 \\ ChargeSwitch & 253 & 1526.3 & 0.63 & 0.29 \\ gDNA & 281 & 1311.8 & 0.63 & 0.26 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Comparison of DNA extraction kits using Nanodrop measurements for assessment. Extracted DNA (1 \(\upmu\)L) from two samples was measured with an ND-1000 spectrometer. Sample ID refers to identity of samples from the TanAsp collection, 260/280 is a measurement of DNA/protein ratio, and 260/230 is a measure of DNA/contaminant ratio. The preferred kit was chosen on the basis of both better ratios. 30 s (denaturing), 56 \({}^{\circ}\)C for 60 s (annealing), 72 \({}^{\circ}\)C for 60 s (elongation) and final extension at 72 \({}^{\circ}\)C for 7 min. To judge the quality of a PCR run the products were visualized under UV light after gel electrophoresis in a 1% agarose gel containing GelRed(r) (Biotium, Inc., Fremont, CA, USA) (3 \(\upmu\)L/100 mL agarose gel) at 140 volts for 30 min. The five fungal-specific primers (Figure 1a, point 4) were tested according to the initial protocol settings above and optimization adjustments were performed mainly by the choice of fungal ribosomal primers specific to the ITS region they targeted. The extended PCRs step tested the binding of the extension (Figure 1a, point 5). Four extension primers were designed to test how well the extension adhered to the fungal primer pairs (Figure 1a, point 4). We did not include any primer pairs targeted for the ITS1 region as their respective fungal-specific primer pairs failed to amplify the ITS1 region. We included extended primer pairs responsible for the entire ITS and for the subregion ITS2 (Figure 2). To optimize for a lower amount of template DNA, forward and reverse primers (0.5 \(\upmu\)L) were used when no bands were found. The annealing temperature was adjusted back and forth from the calculated temperatures to obtain the best bands and cycle number was increased to increase band intensity during PCR improvement (for details and values, please refer to Table 2 and Figure 2). Figure 2: Schematic diagram showing the sequences of primers and general sequential steps for fungal amplicon library preparation. (1) HTS (Illumina MiSeq) sequencing can detect genetic material from many kinds of organisms found in an environmental sample. We were studying the leaves of  
aspen and were interested in detecting the composition of the leaves' fungal microbiome. (2) After freeze drying and grinding leaves, the DNA was extracted using kits and its quality and quantity were measured to check the purity. (3) Fungal DNA can be distinguished from DNA from other organisms through specific and highly conserved regions in the ribosomal genes referred to as internal transcribed spacer (ITS) regions, which are not shared by plants or bacteria.