DNeasy PowerLyzer PowerSoil Kit (100)

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Publication protocol

DNA was extracted with a DNeasy Power Lyzer Power Soil Kit-100 (Qiagen, Hilden, Germany) (Cat. no. 12888-100) for each sample, following the manufacturer’s procedure. DNA extracts were then quantified by an Invitrogen™ Qubit® dsDNA High Sensitivity (HS) Assay Kit (Life Technologies, Eugene, OR, USA). the Australian Genome Research Facility (University of Adelaide, Plant Genomics Centre, Hartley Grove, Urrbrae, SA 5064, Australia) performed the PCR amplification and sequencing. For bacterial identification, the V1-V3 16S rRNA region was amplified by using primers 27F (5′AGAGTTTGATCMTGGCTCAG-3′) and 519R (3′ GWATTACCGCGGCKGCTG-5′) [55]. For the fungi, the ITS region of RNA operon was amplified by using the fungal-specific forward primer ITS1F (5’-CTTGGTCATTTAGAGGAAGTAA-3’) and the reverse primer ITS2 (5’-GCTGCGTTCTTCATCGATGC-3’) [112,113,114]. Reactions contained 1X AmpliTaq Gold 360 mastermix (Life Technologies, Eugene, OR, USA) and 0.20 µM of each forward and reverse primer with 25 µL of DNA. The cycling conditions of the PCR consisted of denaturation at 95 °C for 7 min, 35 cycles of 94 °C for 45 s, 50 °C for 60 s, and 72 °C for 60 s, as well as a final extension of 72 °C for 7 min. A secondary PCR was used to adhere sequencing adaptors and indexes to the amplicons. Primerstar max DNA Polymerase was used for secondary PCR amplicon generation from Takara Bio Inc., Shiga, Japan (Cat. No. #R045Q). The resulting amplicons were measured by fluorimeter (Invitrogen Picogreen, Thermo Fisher Scientific, NSW, Australia) and normalized [115]. The equimolar amounts of each sample were pooled and quantified by qPCR prior to sequencing (Kapa qPCR Library Quantification kit, Roche, Basel, Switzerland). The resulting amplicon library was then sequenced on the Illumina MiSeq platform (San Diego, CA, USA), with 2 × 300 base pairs paired-end chemistry [116].

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