Dna prep

Approximately 200mg of stool was weighed out and diluted to 1:6 with SM Buffer then was vortexed at max speed for three minutes to fully homogenize. Samples were centrifuged at 4° for five minutes at 20,000g. The centrifuged pellet (metagenomics) was taken through Qiagen’s DNeasy PowerSoil Pro Kit following kit protocol recommendations and then followed by Illumina’s DNA prep for library builds and sequenced on a NextSeq 2000 (2×150). The supernatant (viral) was filtered by a 0.20μm filter and then processed by a VLP enrichment before extractions. A mastermix of Benzonase (2μl), Baseline Zero 10x Buffer (100μ), and Baseline Zero DNASE (4μl) was added to each sample and heated for 37° for one hour. After the heat treatment 1mL was transferred into bioMérieux’s EMAG for total nucleic acid (TNA) extraction. The TNA was amplified with GenomiPhi V2 (GE Healthcare) before moving into library builds with DNA prep (Illumina) and sequencing with a NextSeq 2000 (2×150). Controls of SM Buffer spiked with lambdavirus DNA were used to assess cross-sample contamination during amplification and sequencing.

Whole-genome sequencing of SARS-CoV-2 was performed following the ARTIC Network protocol¹ with the V3 primer pools (Integrated DNA Technologies, IDT, IA, United States) for complete genome multiplex, overlapping amplification. Library preparation was performed with KAPA HyperPrep Kit (Roche Applied Science, Basilea, Switzerland) or Illumina DNA Prep (Illumina, CA, United States) depending on the product’s availability. Briefly, cDNA synthesis was performed with SuperScript IV reverse transcriptase (Invitrogen) and further PCR amplification for the two pools of ARTIC V3 primers with Q5 Hot Start High-Fidelity DNA Polymerase (New England BioLabs, MA, United States). PCR products from each sample were individually indexed using the SeqCap Adapter Kit A/B (Nimblegen, Roche, CA, United States) when using KAPA HyperPrep; or IDT^® for Illumina^® DNA/RNA UD Indexes Set A-D (384 IDX) (Illumina, CA, United States) when using Illumina DNA Prep. Finally, the products were added to the final pooled library tube, which, together with a 5% of PhiX internal DNA control (PhiX V3, Illumina, CA, United States), were loaded in a MiSeq Reagent Kit (600 cycles) v3 (Illumina, CA, United States) and sequenced using the MiSeq platform (Illumina, CA, United States).

To obtain concentrated high-molecular-weight genomic DNA, about 1 cm² of mycelium was taken and ground in liquid nitrogen with a pre-cooled pistil. Genomic DNA was isolated using the MagAttract HMW DNA Kit following the manufacturer’s protocol and subsequent purification steps with magnetic beads (HMW genomic DNA from fresh or frozen tissue protocol), resulting in a total yield of 1 µg. The final concentration was 9.53 ng/µL, measured with a NanoDrop 1000 spectrophotometer (Peqlab Biotechnologie, GmbH, Erlangen, Germany) and Qubit 4 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) using 1X dsDNA HS Assay Kits. DNA extraction, PCR amplification and Sanger sequencing (using nrITS primers) were performed to evaluate possible contamination and confirm the cultures’ identities. First, a paired-end library was constructed using Illumina DNA Prep (earlier known as Nextera DNA Flex Library Prep) and sequenced on NovaSeq 6000 (NovaSeq SP 150 bp Paired-end Flow Cell, Illumina) at the Biomedical Sequencing Facility (BSF, Vienna, Austria). A total concentration of 0.2 µg was used for Illumina library preparation. To supplement the Illumina data, we prepared several Oxford Nanopore libraries using the SQK-LSK109 kit and sequenced them on a MinION sequencer using R9.4.1 flow cells (altogether, four runs were conducted). The total yield of DNA was in the range of 0.12–0.31 µg.