Ampure xp purification kit

To avoid the accumulation of single nucleotide variants during the passage process, viral DNA for MGI platform sequencing was extracted directly from specific tissues. The first-generation culture of porcine bone macrophages was used to prepare extracellular virions for Nanopore sequencing. The QIAamp DNA Mini Kit (51304, Qiagen, Germany) was used for extraction, and the extracted viral DNA was purified using the AMPure XP Purification Kit (A63880, Beckman Coulter, USA). The quality and concentration were evaluated using Nanodrop Lite (ND-LITE-PR, ThermoFisher, USA) and Qubit 4.0 Fluorometer (Q33238, Invitrogen, USA). All these procedures were performed according to the manufacturer's instructions.

Genome-spanning primers from the designed primer panel were diluted to a concentration of 10 pmol/mL and combined into six pools. Every pool included every 6th primer in the list; each pool included 23 primer pairs. Hot-start multiplex amplification reactions were performed in a 25 μL total volume containing 2 μL of cDNA, 0.1 μM of each primer, and 12.5 μL of 2× BioMaster HS-Taq PCR mix (Biolabmix, Novosibirsk, Russia). The following thermal cycling parameters were used: 95 °C for 3 min; 35 cycles (93 °C for 10 s, 57 °C for 30 s, 72 °C for 30 s); and a final extension (72 °C for 5 min). Reactions were performed in a C1000 Touch thermocycler (Bio-Rad, Hercules, CA, USA). Products were analyzed by 2.0% agarose gel electrophoresis in the presence of ethidium bromide. Amplified fragments were mixed, then cleaned by the AMPure XP Purification Kit (Beckman Coulter, UK) according to the manufacturer’s instructions (1:1 sample:beads ratio). Concentrations of the fragment mixes were measured with a Qubit 4.0 fluorimeter (Invitrogen, Waltham, MA, USA) using the dsDNA HS Assay Kit (Invitrogen, Waltham, MA, USA) and then used for library preparation.

Agencourt^® AMPure XP purification kit (Beckman Coulter, Brea, USA) was used for purification of the samples as indicated by the manufacturer. The resulting product was re-purified through DNA Clean & Concentrator (Zymo Research) commercial kit. 1% agarose gel electrophoresis was performed for validation of PCR products.

For all selected isolates, all 8 gene segments (HA, neuraminidase [NA], nucleoprotein [NP], polymerase basic 1 [PB1] and 2 [PB2], polymerase acidic [PA], matrix [M], and nonstructural [NS]) were sequenced by using a next-generation sequencing strategy for influenza A virus sequencing with the Ion PGM System and PathAmp FluA Reagents (Life Technologies, Carlsbad, CA, USA). Viral RNA extraction was performed by using the QIAamp Viral RNA Mini Kit (QIAGEN, Hilden, Germany). Reverse transcription PCR amplification of all 8 gene segments were performed by using PathAmp Flu A Preamplification Reagents (Life Technologies). Amplicons were purified and quantitated by using the Ampure XP purification kit (Beckman, Brea, CA, USA). The genomic libraries were prepared with the Ion Xpress Plus Fragment Library Kit (Life Technologies). Enzymatic fragmentation was used for the 200-bp read protocol with a 10-min incubation time. Samples were assigned barcodes by using the Ion Xpress Barcode Adapters 1–32 Kit (Life Technologies). Automated template preparation was performed by using the Ion OneTouch 2 System (Ion PGM Template OT2 200 Kit; Life Technologies). Final sequencing was performed with the Ion PGM Sequencing 200 Kit v2 (Life Technologies) using the Ion 316 Chip V2.

To create NGS libraries, viral RNA was subjected to reverse transcription and subsequent PCR enrichment. Reverse transcription was performed using random hexamers with the Reverta L Kit (AmpliSens, Russia) following the manufacturer's protocol. Samples (cDNA) were stored at -20°C until amplification. To obtain a near complete genome sequences of SARS-CoV-2 (excluding the 5′ and 3′ ends), a total of 138 primer pairs covering the entire genome were applied according to a previously described multiplex PCR protocol [9 ]. Reactions were performed in a total volume of 25 μl containing 2 μl cDNA, 0.1 μM of each primer, and 12.5 μl 2 × BioMaster HS-Taq PCR mix (Biolabmix, Novosibirsk, Russia). PCR were performed with following parameters: 95 °C for 3 min; 40 cycles (93 °C for 10 s, 57 °C for 30 s, 72 °C for 30 s); a final extension (72 °C for 5 min). Reactions were performed in a C1000 Touch thermal cycler (Bio-Rad, USA). The products were analyzed by electrophoresis on a 2.0% agarose gel in the presence of ethidium bromide. Reaction products were purified using the AMPure XP purification kit (Beckman Coulter, UK) in 1:1 ratio according to the manufacturer's instructions and equimolary pooled. The concentration of the PCR-fragment mixture was measured using the dsDNA HS Assay Kit (Invitrogen, USA) with a Qubit 4.0 Fluorimeter (Invitrogen, USA) and used for library preparation.

Soil grains, rocks, and organic matter (plant roots and leaves) were manually removed, and 1 mg of the sampled biocrust was used for DNA extraction with MoBio Powersoil kit (Laboratories Inc., Carlsbad, CA, USA) according to the manufacturer’s instructions. The region V3-V4 of 16S rRNA gene (420 bp) was amplified with CYA 359 and 781a/b primers as described in Nübel et al. (1997) , with an overhang Illumina adapter included in the primers. The PCR reaction contained 10 ng of eDNA, 0.2 μM of each primer, and 1x KAPA HiFi HotStart Ready Mix (KAPA Biosystems) to 25 μl of final volume. The PCR product from the samples 1–3, 4–6, and 7–10 of each transect were pooled and purified using AMPure XP purification kit (Beckman Coulter Inc., Brea, CA, USA). Afterwards, Illumina sequencing adapters and dual-index barcodes were added to the amplicon target using the Nextera XT Index Kit (Illumina, USA), according to the manufacturer’s instructions. The product was purified using AMPure XP purification kit, quantified with Qubit Fluorometric Quantitation (Thermofisher/Life Technology, USA). The samples were normalized and, then, pooled in an equimolar fashion. The preparation of the samples followed the Illumina guidelines for sequencing in a MiSeq platform (Illumina) available at Center of Nuclear Energy and Agriculture (ESALQ/USP) and using MiSeq Reagent kit v3. 2 × 300 cycle.

Saliva DNA was extracted using a two-step protocol, including the sample preprocessing with lysozyme lysis (lysozyme from chicken egg white; Sigma-Aldrich) and bead beating and the TIANamp blood DNA kit (Beijing, China). The 16S rRNA amplicon library was amplified with 341F/805R primers (CCTACGGGNGGCWGCAG, GACTACHVGGGTATCTAATCC; V3-4) (41 (link), 42 (link)). Samples were amplified with 20 cycles of a program with 30 s at 98°C for melting, 30 s at 60°C, and 30 s at 72°C using Hi Fi Kappa HotStart Ready Mix (Roche). Samples were barcoded in a second PCR step (41 (link)). DNA cleanup was performed using an Agencourt AMPure XP purification kit. DNA volume and purity were measured on an Agilent 2100 Bioanalyzer system and real-time PCR. Sequencing was performed at Beijing Genome Institute (BGI) on an Illumina MiSeq using a 2× 300-bp paired-end strategy.