Hifi hotstart readymix

PCR was performed using universal primers flanking the variable 4 (V4) region of the bacterial 16S rRNA gene [76 (link)]. Genomic DNA samples were amplified in duplicate. Each reaction contained 10–30 ng genomic DNA, 10 μM each primer, 12.5 μl 2x HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA), and water to a final reaction volume of 25 μl. PCR was carried out under the following conditions: initial denaturation for 3 min at 95°C, followed by 25 cycles of denaturation for 30 s at 95°C, annealing for 30 s at 55°C and elongation for 30 s at 72°C, and a final elongation step for 5 min at 72°C. PCR products were purified with the QIAquick 96-well PCR Purification Kit (Qiagen, Germantown, MD, USA) and quantified using Qubit dsDNA HS Assay kit (Invitrogen, Oregon, USA). Samples were equimolar pooled and sequenced by the University of Wisconsin–Madison Biotechnology Center with the MiSeq 2x250 v2 kit (Illumina, San Diego, CA, USA) using custom sequencing primers.

PCR was performed using universal primers flanking the variable 4 (V4)
region of the bacterial 16S rRNA gene^{39 (link)}. Genomic DNA samples were amplified in duplicate. Each
reaction contained 25 ng genomic DNA, 10 μM of each uniquely barcoded
primer, 12.5 μl 2x HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington,
MA, USA), and water to a final reaction volume of 25 μl. PCR was carried
out under the following conditions: initial denaturation for 3 min at
95°C, followed by 20 cycles of denaturation for 30 s at 95°C,
annealing for 30 s at 55°C and elongation for 30 s at 72°C, and a
final elongation step for 5 min at 72°C. PCR products were purified with
the QIAquick 96-well PCR Purification Kit and quantified using the Qubit dsDNA
HS Assay kit (Invitrogen, Oregon, USA). Samples were equimolar pooled and
sequenced by the University of Wisconsin–Madison Biotechnology Center
with the MiSeq 2×250 v2 kit (Illumina, San Diego, CA, USA) using custom
sequencing primers.

The rhizosphere DNA was extracted from 0.5 g sample using the DNeasy PowerSoil Kit (Qiagen, Hilden, Germany) and DNA samples were randomized across plates. The bacterial V4 region of the 16S rRNA gene was amplified using the protocol described by Lundberg et al. (2013) (link). The universal primer pair 515F and 806R was used to generate bacterial-derived 16S rRNA amplicons. PNA PCR clamps were used to reduce host organelle contamination. The fungal ITS2 region was amplified using the universal primers ITS3/KYO2 and ITS4 (Toju et al., 2012 (link)). All primers were modified to include Illumina adapters¹. Each 25 μl reaction contained 12.5 μl of HiFi HotStart Ready Mix (KAPA Biosystems, Woburn, MA, United States), 1.0 μl of each primer (10 μM), 2.5 μl of DNA template (5 ng/μl), and 8.0 μl PCR-grade water. PCR amplifications (performed in triplicate for each sample) consisted of a 3 min denaturation at 95°C; 25 cycles of 30 s at 95°C, 30 s at 55°C and 30 s at 72°C; and 5 min at 72°C. Samples were cleaned using the AMPure beads XP purification system (Beckman Coulter, United Kingdom) and sequenced on the Illumina MiSeq platform at the Fundación FISABIO (Valencia, Spain) facility using a 2 × 300 nucleotide paired reads protocol.

After expert pathologist assessment, solid tumor genomic DNA was isolated from formalin-fixed, paraffin-embedded tumor samples. DNA libraries were prepared using the KAPA Hyper Prep Kit, hybridized to the xT probe set, and amplified with the KAPA HiFi HotStart ReadyMix. The amplified target-captured DNA tumor libraries were sequenced to an average target depth of 500 × on an Illumina HiSeq 4000 using the Tempus xT (Tempus labs Chicago, IL, USA) gene panel. The panel analyzes single nucleotide variants (SNVs), indels, and copy number variants in 596 genes and genomic rearrangements in 21 genes with an average coverage of 500x^{28 (link)}. Variants were called using Freebayes (version 1.0.2). Clinically relevant mutations were identified using the Cancer Genome Interpretor^{29 (link)} and ClinVar^{30 (link)}. Data was graphed using Microsoft Excel 2016.

The genomic DNA of the plant tubers was extracted using CTAB [56 (link)]. Genomic DNA from the surrounding soils was extracted using a commercial DNA extraction kit (PowerSoil® DNA Isolation Kit, USA). The DNA quality was monitored using 0.8% agarose gel electrophoresis, and the extracted DNA was diluted to a concentration of 1 ng/μL and stored at − 20 °C until further processing. The diluted DNA was used as a template for the PCR amplification of fungal ITS genes with the barcoded primers and a HiFi Hot Start Ready Mix (KAPA, USA).
The ITS region of the fungi was amplified using the primers 1743F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and 2043R (5′-GCTGCGTTCTTCATCGATGC-3′) [57 (link)]. The 5′-barcoded amplicons were generated by PCR under conditions of 2 min at 95 °C, followed by 35 cycles at 95 °C for 30 s, annealing at 55 °C for 1 min, extension at 72 °C for 1 min, and a final extension at 72 °C for 10 min. Amplicon quality was visualized using gel electrophoresis, purified with AMPure XP beads (Agencourt) and amplified for another round of PCR. After purification with the AMPure XP beads again, the final amplicon was quantified using a Qubit dsDNA assay kit. Equal amounts of purified amplicon were pooled for subsequent sequencing at the Shanghai OE Biotech. Co., Ltd. (Shanghai,China).

A fraction of each homogenized gut was used for total DNA extraction using the MasterPure^TM Complete DNA & RNA Purification Kit (Epicenter, Madison, WI, United States) according to the manufacturer’s instructions. Purified DNA was quantified in QUBIT and 10 ng/ul were used for amplification and sequencing of the 16S rRNA gene from 77 samples. The amplicon sequencing protocol targets the 16S gene V3 and V4 regions (459 bp), with the primers designed surrounding conserved regions (Klindworth et al., 2013 (link)). Following the Illumina amplicon libraries protocol, DNA amplicon libraries were generated using a limited cycle PCR: initial denaturation at 95°C for 3 min, followed by 25 cycles of annealing (95°C 30 s, 55°C 30 s, 72°C 30 s), and extension at 72°C for 5 min, using a KAPA HiFi HotStart ReadyMix (KK2602). Then Illumina sequencing adaptors and dual-index barcodes (Nextera XT index kit v2, FC-131-2001) were added to the amplicon. Libraries were then normalized and pooled prior to sequencing. The pool containing indexed amplicons was then loaded onto the MiSeq reagent cartridge v3 (MS-102-3003) spiked with 10% PhiX control to improve base calling during sequencing, as recommended by Illumina for amplicon sequencing. Sequencing was conducted using a 2 × 300-pb paired-end run on an Illumina MiSeq sequencing system.