Longamp hot start taq 2x master mix

16S rRNA amplicon sequencing was performed on a MinION nanopore sequencer (Oxford Nanopore Technologies, Oxford, UK). The amplicon library was prepared using the 16S Barcoding Kit 1–24 (SQK-16S024, Oxford Nanopore Technologies, Oxford, UK). For the PCR amplification and barcoding, 15 ​ng of template DNA extracted from fecal samples, and 30 ​ng in the case of saliva, were added to the LongAmp Hot Start Taq 2X Master Mix (New England Biolabs, Ipswich, MA). Initial denaturation at 95 ​°C was followed by 35 cycles of 20 ​s at 95 ​°C, 30 ​s at 55 ​°C, 2 ​min at 65 ​°C, and a final extension step of 5 ​min at 65 ​°C. The barcoded amplicons were purified using the AMPure XP beads (Beckman Coulter, Brea, CA) as per Nanopore's instructions. Samples were then quantified using a Qubit fluorometer (Life Technologies, Carlsbad, CA) and pooled in an equimolar ratio to a total of 50–100 ​ng in 10 ​μl. The pooled library was then loaded into an R9.4.1 flow cell and run per the manufacturer's instructions. MINKNOW software 19.12.5 was used for data acquisition. The provided primer set includes a recently noted issue with the standard ONT forward primer, which contains three mismatching bases to the family Bifidobacteriaceae and thus fails to amplify microbes of these taxa (Fujiyoshi et al., 2020 (link)).

Nearly 200 grams of 72 samples obtained from potato tuber-sphere at harvest or after one-month post-harvest storage, from all the individual collection sites (Table S10), was used for the microbial mapping of the two different regions. High quality DNA was isolated with the DNeasy PowerSoil Pro Kit (QIAGEN, Carlsbad, USA), following the manufacturer’s instructions and stored at −80°C. Amplification of the 16S rRNA gene was performed using an Applied Biosystems® QuantStudio® 5 Real-Time PCR System (Thermo Fischer Scientific, Waltham, MA, USA), using a LongAmp Hot Start Taq 2x Master Mix (M0533S, New England Biolabs), and 16S barcoded primers.
The 16S Barcoding Kit 1-24 (SQK-16S024, Oxford Nanopore Technologies, UK) was used for sequencing the 16S ribosomal gene and creating the libraries. PCR products were purified with Agecount AMPure XP beads (Beckman Coulter, USA), whilst the quantification was performed using Qubit 4 Fluorometer and the dsDNA HS Assay Kit (Thermo Fisher Scientific, USA). The 72 libraries were created in accordance with the manufacturer’s instructions and loaded on a MinION R9.4.1 flow cell (FLO-MIN106) on the MinION Mk1C (Oxford Nanopore Technologies, UK). For data acquisition, MINKNOW software ver. 1.11.5 (Oxford Nanopore Technologies) was employed.

Genotyping PCR was performed using genomic DNA extracted from the toe clipping with the primers listed in Supplemental Materials. For Sanger sequencing, PCR products were first gel-purified and cloned into the pCRII Topo vector (Thermo Fisher Scientific K460001) and sequenced with T7 or SP6 primers. Long-range PCR was performed using LongAmp Hot Start Taq 2X Master Mix (NEB M0533S) to examine genomic DNA that was purified by a Monarch Genomic DNA Purification kit (NEB T3010). The large amplicons were gel-purified and cloned into the pCR-XL-2-TOPO vector (Thermo Fisher Scientific K8050-10). The top off-targeting sites were predicted by Cas-OFFinder (Bae et al. 2014 (link)).