DNA extraction, PCR and Sequencing
Isolation of genomic DNA from the samples was carried out using the NucleoSpin Plant Kit (Machery-Nagel, Germany) following the manufacturer’s protocol. The resulting DNA-extracts were stored at -20 °C. DNA concentrations were determined using the Quantus Fluorometer (Promega, USA) according to the manufacturer’s protocol. A total of 21 samples, which were amplified using the 3 different primer sets (total of 63 rDNA libraries) were processed and compared in this study (Supplementary Table S1). For Illumina Sequencing, a fragment of the 18S rDNA containing the hypervariable V4 region was amplified with the primer sets (i) Reuk454FWD1 (5’-CCAGCASCYGCGGTAATTCC-3’) and ReukREV3 (5’- ACTTTCGTTCTTGAT-3’) (Stoeck et al., 2010, hereafter referred to as Stoeck), (ii) Wolf938 and (iii) Wolf964. The mock community sample was also amplified with the primer set Reuk454FWD1 and V4r (Bradley et al., 2016).
All PCR reactions had a final volume of 25 µL containing 12.5 µl of KAPA HIFI Mix (Kapa Biosystems, Roche, Germany), 5 µl of each primer [1 µmol/L] and 2.5 µl DNA-template [~5ng]. The DNA-template was a mix of equal volumes of genomic DNA isolated from the three different filter fractions. PCR amplification was performed in a thermal cycler (Eppendorf, Germany) with an initial denaturation (95 °C, 3 min) followed by 25 cycles of denaturation (95 °C, 30 sec), annealing (55 °C, 30 sec), and extension (72 °C, 30 sec) with a single final extension (72 °C, 5 min). The PCR products were purified from an agarose gel 1% [w/v] with the AMPure XP PCR purification kit (Beckman Coulter, Ing., USA) according to the manufacturer’s protocol. Subsequent to purification the DNA concentrations in the samples was determined using the Quantus Fluorometer (Promega, USA). Subsequently, indices and sequencing adapters of the Nextera XT Index Kit (Illumina, USA) were attached in the course of the Index PCR. All PCRs had a final volume of 50 µL and contained 25 µl of KAPA HIFI Mix (Kapa Biosystems, Roche, Germany), 5 µl of each Nextera XT Index Primer [1 µmol/L], 5 µl DNA-template [~5ng] and 10 µl PCR grade water. PCR amplification was performed in a thermal cycler (Eppendorf, Germany) with an initial denaturation (95 °C, 3 min) followed by 8 cycles of denaturation (95 °C, 30 sec), annealing (55 °C, 30 sec), and extension (72 °C, 30 sec) with a single final extension (72 °C, 5 min). Prior quantification of the PCR products with the Quantus Fluorometer (Promega, USA) for sequencing with the MiSeq-Sequencer (Illumina, USA), the final library was cleaned up using the AMPure XP PCR purification kit (Beckman Coulter, Ing., USA). Sequencing of the DNA-fragments was carried out with the MiSeq Reagent Kit V3 (2 x 300 bp) according to the manufacturer’s protocol (Illumina, USA). Sequences generated in this study were deposited at the European Nucleotide Archive (ENA) with the accession number xxx (accession number will be provided subsequent to acceptance of the manuscript ).