title: DNA amplicon sequencing of arbuscular mycorrhizal fungi from Arctic soils using long-read SSU-LSU primers
design: Qiagen DNeasy PowerSoil Pro extraction kit was used to isolate environmental DNA from the processed soil samples following the manufacturers protocol. DNA was extracted from approximately 250 mg of soil from each sample. The samples were then checked using Qubit dsDNA High Sensitivity Assays for the presence and concentration of DNA in the sample prior to PCR and stored in -20 C until further analyses. Two sets of PCRs were performed using two pairs of primers targeting different regions of the fungal genome. ITS1, ITS2, and partial LSU regions were amplified using the ITS1ngsLR5 primer pair (ITS1m: 5-TCCGTAGGTGAACCTGC-3 Tedersoo et al. (2015); LR5: 5-TCCTGAGGGAAACTTCG-3 Vilgalys and Hester (1990)) to broadly capture fungal groups (Eshghi Sahraei et al. 2022). These ITS-LSU region primers may have difficulty targeting some groups of saprotrophic and pathogenic fungi, as well as AM fungi (Tedersoo et al. 2022), although these groups (aside from AM fungi below) were not of interest for this study. Partial SSU region was amplified using the SSU515FngsAML2 primer pair (SSU515Fngs: 5-GCCAGCAACCGCGGTAA-3 Tedersoo et al. (2015); AML2: 5-CCCAAACACTTTGGTTTCC-3 Lee et al. (2008)) to target AM fungi specifically. A reaction volume of 50 L was used for PCR with 5 L each of template DNA (approximately 1-1.5 g of DNA per sample), forward and reverse barcoded primers with sample specific tags (Table S3, Tedersoo et al. 2015, Eshghi Sahraei et al. 2022) and 0.5 L of Phusion High-Fidelity DNA polymerase. A negative control with no template DNA was included with each PCR run. Thermocycling conditions for the ITS1ngsLR5 region were an initial denaturation at 98 C for 30 s followed by 25 cycles of denaturation at 98 C for 10 s, annealing at 59 C for 45 s and extension at 72 C for 45 s, with a final extension for 10 minutes after the final cycle. Thermocycling conditions for the SSU515FngsAML2 primer pair were an initial denaturation at 98 C for 30 s followed by 30 cycles of denaturation for 10 s, annealing at 58 C for 30 s and extension at 72 C for 30 s and a final extension for 7 minutes after the final cycle. A total of 232 PCR products were cleaned using Agencourt AMpure XP magbeads (Beckman Coulter, Brea, CA, USA) and quantified using Qubit dsDNA High Sensitivity Assays prior to pooling for equimolar concentrations. A maximum volume of 48 L was used for samples with too low concentration. Library preparation and sequencing was performed by Uppsala Genome Centre (UGC, Science for Life Laboratory, Dept. of Immunology, Genetics and Pathology, Uppsala University, BMC, Box 815, SE-752 37 UPPSALA) in Uppsala, Sweden using two SMRT cells on the Sequel platform (Pacific Biosciences, Menlo Park, CA, USA).