title: Amplicon sequencing of soil bacteria: temperate agroforestry
design: Soil bacteria were amplified using the primer pair 341F (5-CCTACGGGNGGCWGCAG-3) / 785R (5-GACTACHVGGG TATCTAAKCC-3) (Klindworth et al. 2013). To overcome PCR inhibition, DNA extracts from topsoil and subsoil samples were diluted 1:50 (v/v) in double-distilled water prior to PCR. The composition of the master mix and the thermocycling conditions for the PCR and preparation of sequencing library were identical to those of Bednar et al. (2023). To verify success of amplification, PCR products were assessed on 1% (w/v) agarose gels stained with SYBR Green I solution (Thermo Fisher Scientific GmbH, Dreieich, Germany). Sequencing of the libraries was done at LGC Genomics (Berlin, Germany) on an Illumina MiSeq (V3 chemistry, 2 300 bp) (Illumina, Inc., San Diego, CA, USA). Prior to sequencing, a second amplification with standard i7- and i5- sequencing adapters as well as multiplexing was performed.