## 2 Materials and Methods
### Primers, PCRs, and Gel Electrophoresis
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Purpose** & **Primers** & **Template DNA** & **Settings** & **Outcome** & **Decision/Selection** \\ \hline \multirow{7}{*}{Fungal PCR for ITS-selection} & ITS1 (f) + & 1. 1 \(\mu\)L & 1. 56/30 * & 1. SB with desired DF (a) & 1. Pp failed to bind to fungal ITS region. \\  & ITS4 (r) & 1. 1 \(\mu\)L & 1. 56/30 & 1. NB & 1. Pp failed to bind to ITS. \\  & ITS51F (f) + ITS4 (r) & 1. 1 \(\mu\)L & 2. 57/30 & 2. SB with desired DF (b) & 2. Successful amplification of ITS. \\  & ITS51 (f) + & 1. 1 \(\mu\)L & 1. 56/30 & 1. NB & 1. Pp failed to bind to ITS1. \\  & ITS52 (r) & 1. 1 \(\mu\)L & 2. 57/30 & 2. NB & 2. Pp failed to bind to ITS1. \\  & ITS3 (f) + & 1. 1 \(\mu\)L & 1. 57/30 & 1. SB with desired DF (c) & 1. Pp successfully amplified ITS2. \\  & ITS4 (r) & 1. 1 \(\mu\)L & 1. 56/30 & 1. FB (d) & \\ \hline \multirow{7}{*}{Ext. PCR for ITS ampl.} & & 1. 1 \(\mu\)L & 1. 62/30 & 1. FB (d) & \\  & & 2. 1 \(\mu\)L & 1. 62/30 & 2. FB with PD & \\  & & 3. 1 \(\mu\)L & 2. 60/30 & 3. FB with undesired DF (e) & \\  & & 4. 1 and 0.5 \(\mu\)L & 4. 62/30 & 4. FB with undesired DF & \\  & Ext-ITS1F (f) + & 5. 1 and 0.5 \(\mu\)L & 5. 64/30 & 5. FB with undesired DF & \\  & Ext-ITS4 (r) & 6. 1 and 0.5 \(\mu\)L & 6. 66/30 & 6. FB with undesired DF & \\  & & 7. 1 and 0.5 \(\mu\)L & 6. 66/30 & 7. FB with undesired DF & \\  & & 8. 0.5 and 1 \(\mu\)L & 8. 60/30 & 8. SB with desired DF & \\  & & PCR Product & 9. 58/33 & 9. PD with both desired and & \\  & & 9. 1 and 0.5 \(\mu\)L & & undesired DF (f) & \\ \cline{2-5}  & Ext-ITS1 (f) + & 1. 1 \(\mu\)L & 1. 56/30 & 1. NB & 1. Ext. Pp failed to amplify ITS. \\  & Ext-ITS4 (r) & 2. 1 \(\mu\)L & 2. 58/30 & 2. NB & 2. Ext. Pp failed to amplify ITS. \\ \hline \multirow{3}{*}{Index PCR} & \multirow{3}{*}{Index primer pair} & 1. 8 \(\mu\)L of PCR & & 1. Index Pp failed to amplify, \\  & & product of Ext-ITS1F & 1. 55/8 & 1. FB with PD (h) & suggesting absence of ext. region \\  & & (f) + Ext-ITS4 (r) & & in PCR products. \\ \hline \multirow{3}{*}{Ext. PCR} & \multirow{3}{*}{Ext-ITS3 (f) +} & 1. 1 \(\mu\)L & 1. 72/33 & 1. NB (g) & 1. Ext Pg failed to amplify ITS2. \\  & Ext-ITS4 (r) & 2. 1 \(\mu\)L & 2. 70/33 & 2. FB (g) & 2. Ext Pg failed to amplify ITS2. \\ \cline{1-1}  & & 3. 1 \(\mu\)L & 3. 57/33 & 3. SB with desired DF (g) & 3. Set PP amplified ITS2. \\ \hline \multirow{3}{*}{Index PCR for ITS2 ampl.} & \multirow{3}{*}{Index primers} & 1. 8 \(\mu\)L PCR product & & 1. Index Pp successfully amplified \\ \cline{1-1}  & & of Ext-ITS3 (f) + & 1. 55/8 & 1. Desired difference between & 1. Index Pp failed to amplify, \\ \cline{1-1}  & & Ext-ITS4 (r) & & index PCR and Ext. PCR(i). & \\ \hline \multirow{7}{*}{1. 56/30 means 1/1 is the serial number of the repeated step, 56/ is the annealing temperature and  ‘30/ is the number of PCR cycles. Abbreviations: DF = DNA Fragment; FB = faint band; Ext. = extended; NB = no band; SB = strong band; PD = primer dimers, Pp = primer pairs; ampl = amplification.} \\ \cline{1-1}  & & & & \\ \end{tabular}
\end{table} 
Table 2: An overview of the optimization procedure for preparation of aspen leaf metagenomics samples for HTS sequencing. Letters (a–i) refer to the resulting PCR gels as shown in Figure 3. Template DNA refer to volume of sample DNA extract. Settings = PCR settings indicated as “annealing temperature “C”/“number of cycles”. index primers (Figure 1a, point 6). The same protocol was followed as described above for fungal PCR, although extended PCR products replaced template DNA, and index primers replaced forward and reverse primers. Following the SciLife protocol, up to 8 \(\upmu\)L extended PCR product (equivalent to 5 ng of sample) was added to provide a total volume of 20 \(\upmu\)L. The PCR protocol followed SciLifelab's recommendations: initial denaturation at 98 \({}^{\circ}\)C for 2 min followed by 8 cycles of 98 \({}^{\circ}\)C for 20 s (denaturing), 55 \({}^{\circ}\)C for 20 s (annealing), 72 \({}^{\circ}\)C for 15 s (elongation) and final extension at 72 \({}^{\circ}\)C for 2 min.