Qiaxcel advanced system

All bisulfite-modified DNA samples were amplified using separate multiplex or simplex PCRs. The assay designed using Assay Design Service (ADS) software (v1.0.6, QIAGEN, Hilden, Germany) for genes or regions. The PCRs included 0.5 units of Qiagen HotStarTaq, 0.2 µM primers, and 3 µL of bisulfite-treated DNA in a 20 µL reaction mixture. QIAxcel Advanced System (v1.3.0, QIAGEN, Hilden, Germany) were used to quantify the PCR products. Meanwhile, PCR products from the same sample were pooled and purified using QIAquick PCR Purification Kit columns (Qiagen). Libraries were prepared using a KAPA Library Preparation Kit for Ion Torrent Platforms (cat# KK8310) and Ion Xpress™ Barcode Adapters (Thermo Fisher, Waltham, MA, USA). Next, the libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and quantified using the Qiagen QIAxcel Advanced System. The barcoded samples were pooled in an equimolar fashion before template preparation, and enrichment was performed on an Ion Chef™ system (Thermo Fisher, Waltham, MA, USA) using Ion 520™ and Ion 530™ Chef reagents. Next, the enriched, template-positive libraries were sequenced on an Ion S5™ sequencer using Ion 530™ sequencing chips (Thermo Fisher, Waltham, MA, USA).

MinION libraries were prepared from 400 ng of pure plasmid or total genomic DNA using the SQK-RBK004 Rapid Barcoding Kit and sequenced using the FLOW-MIN 106 (R9.4 SpotOn) flow cell according to instructions from ONT. The total genomic DNA and pure plasmid DNA of each strain were barcoded separately. ONT’s MinKNOW software (version v18.03.1) collected raw electronic data as Fast5 read files, and bases were called using ONT’s EPI2ME software. Initial real-time workflows “What Is in My Pot?” (WIMP) were used to confirm bacterial biotypes based on 16S rDNA. Sequence data were collected for 24 h.
Illumina paired-end libraries were prepared from total genomic DNA or pure plasmid DNA using a Nextera DNA Flex library prep kit on an Illumina iSeq 100 instrument and sequenced with 150 bp paired reads according to the manufacturer’s instructions. Quality control of library preparation was performed using the QIAxcel Advanced Systems (Qiagen, Hilden, Germany). Nanopore and Illumina sequencing was performed at the Athens Veterinary Diagnostic Laboratory, University of Georgia, Athens, GA, USA.

The PCR reactions were carried out in a Bio-Rad C1000 Touch © Thermal Cycler (Bio-Rad, Hercules, California USA). Initial PCR’s were performed using four different volumes of target DNA and PCR grade water: 1, 2, 2.5 and 3.5 μL of target DNA and 3.65, 2.65, 2.15 and 1.15 μL of PCR grade water, respectively. The best quality of bands and detection was achieved with 3.5 μL of target DNA. Hence, the PCR components were optimized as follows: 6.25 μL of 2x Qiagen Multiplex Master Mix (Qiagen, Hilden, Germany), 0.1 μL of 5xQ-solution (Qiagen, Hilden, Germany), 0.25 μL of Bovine Serum Albumin (Thermo Scientific, Waltham, MA USA), 1.15 μL of PCR grade water, 1.25 μL of primer mix, and 3.5 μL of DNA, to the total volume of 12.5 μL. The PCR reaction cycle started with an initial denaturation at 95°C for 15 min, followed by 34 cycles of 30 s denaturation at 95°C, 90 s annealing at 60.0°C, 90 s of extension at 72°C and a final extension at 72°C for up to 10 min. The PCR products were visualized using QIAxcel Advanced Systems (Qiagen®).
Primer mixes were created and tested based on the primers target product size, compatibility, sensitivity and specificity towards their target DNA. Step-wise testing was performed to adjust the concentration of primers within each mix to ensure standardized sensitivity of all primer pairs for their target plant DNA.

For this study, we used the 8 microsatellite loci (SS3, SS11, SS16, SS37, SS40, SS43, SS53, and SS54) described by Arranz et al. (2013 (link)). PCR reactions were performed in a 15 μl reaction volume containing 10–100 ng of template DNA, 1 × PCR Buffer, 2 mM of MgCl₂, 0.5 μM of each dNTPs, 0.2 μM of each primer, and 1 U of Taq DNA polymerase (Takara, China). The reaction profile included an initial denaturation at 95°C for 5 min, followed by 36 cycles of initial denaturation at 95°C for 50 s, an annealing temperature of 53°C for 50 s, extension at 72°C for 30 s, and a final extension step at 72°C for 7 min. We analyzed amplified fragments using the QIAxcel DNA High Resolution cartridge and method OM800 on the QIAxcel Advanced System (QIAGEN, Germany). Allele sizing was performed using QIAxcel ScreenGel software version 1.0.2.0 (QIAGEN, Germany) by comparing alleles with a molecular alignment marker (10 and 600 bp, QIAGEN) and size marker (25–500 bp, QIAGEN).