phoD amplicon sequencing of the bulk soil at 60-70 cm depth: Bulk soil at 60-70 cm depth replicate 2
phoD library preparation using the primers phoD_FW and phoD_RW (Eder et al., 1996). Amplification: 94C for 2 min; 94C for 30 s / 57C for 60 s / 72C for 45 s (35 cycles); 72C for 7 min. PCR reaction mixture (total 25 ul); 25 ng of template DNA, 0.5 ul of each primer (10 pmol), 1.5 ul of 3% BSA, 1.0 ul of 50mM MgSO4, 0.5 ul of 10mM dNTP, 2.5 ul of 10 High Fidelity PCR Buffer, 0.2 ul of Platinum Taq DNA Polymerase High Fidelity (5U/ul) (Invitrogen, Germany), an adequate amont of DEPC-treated water. Sample purification: Agencourt AMPure XP extraction kit (Beckman Coulter Company), the ratio of AMPure XP to PCR reaction is adapted to 0.8. Indexing PCR: Nextera XT Index Kit v2 set B, 98C for 5 min; 98C for 10 s / 55C for 30 s / 72C for 30 s (10 cycles); 72C for 5 min. Sample purification: mentioned above. Products are pooled equimolar to a final concentration of 4 nM and sequenced using the MiSeq Reagent kit v3 (600 cycles) for paired end sequencing. Raw sequences were separated from adapters using AdapterRemoval v2.1.7, and reads were trimmed with a minimum read length of 50 bp and a Phred score of 15 (Schubert et al., 2016).