Amplicon sequencing of soil bacteria: temperate agroforestry
Soil bacteria were amplified using the primer pair 341F (5-ACGGGNGGCWGCAG-3) / 785R (5-GACTACHVGGG TATCTAAKCC-3) (Klindworth et al. 2013).  PCR was performed using an Eppendorf Mastercycler EP Gradient S thermocycler (Eppendorf, Hamburg, Germany) in 25-L reaction volumes containing 18.75 L master mix and 6.25 L template DNA or ddH2O for a negative control. The master mix contained ddH2O, buffer (10 mM TrisHCl, 50 mM KCl, 2.0 mM MgCl2, pH 8.3 at 25 C), 100 M of each deoxynucleoside triphosphate (New England Biolabs, Beverly, Massachusetts, USA), 0.5 M of each primer, 1 mg mL1 bovine serum albumin, and 0.03 u L1 Hot Start Taq DNA Polymerase (New England Biolabs, Beverly, Massachusetts, USA). As per Bednar et al. (2023), each primer was a mixture of the primer with (50%) and without (50%) Illumina TruSeq 5-end adapters. We used a touch-up thermocycling protocol (Beule and Karlovsky 2021) to amplify bacteria. The thermocycling conditions were as follows: initial denaturation at 95C for 2 min, 3 touch-up cycles (denaturation: 95C for 20 s, annealing: 50C for 30 s, and elongation: 68C for 60 s), 25 cycles (denaturation: 95C for 20 s, annealing: 58C for 30 s, and elongation: 68C for 60 s). Final elongation was carried out at 68C for 5 min. Amplicons were screened on 1.7% (w/v) agarose gels as described above. Sequencing libraries were subjected to a second amplification with standard i7- and i5- sequencing adapters, multiplexed, and sequenced on an Illumina MiSeq (V3 chemistry, 2  300 bp) (Illumina, Inc., San Diego, CA, USA) at the facilities of LGC Genomics (Berlin, Germany).