Agencourt ampure xp system

The DNA concentration of the samples was measured using the Qubit dsDNA BR Assay kit (Invitrogen). DNA (1–2 µl) was added to Qubit dsDNA BR working solution such that the total volume was 200 µl. The mixture was vortexed for 2–3 s, ensuring that no bubbles formed, and was then incubated for 2–5 min at room temperature prior to the measurement of DNA concentration with a Qubit 2.0 fluorometer (Invitrogen).
DNA purification of amplicons was carried out using the Agencourt AMPure XP system (Beckman Coulter). Room temperature AMPure XP beads were mixed with DNA samples in low DNA-binding microcentrifuge tubes (Eppendorf), after which the mixtures were incubated for 5 min at room temperature. The tubes were then placed next to the magnets on the magnet rack to sit until all the beads were pulled towards the magnet. The supernatant was then removed, after which the beads were washed twice with 1 ml of 80 % ethanol while the tubes were still on the rack. After the supernatant was removed, the beads were allowed to air dry for 5 min on the magnet to allow evaporation of excess ethanol. The tubes were removed from the magnets, and DNA was eluted with EB buffer (Qiagen) The DNA samples were quantified after purification.

The 1D Strand switching cDNA by ligation protocol (Version: SSE_9011_v108_revS_18Oct2016) from the ONT was used for sequencing HSV-1 cDNAs on the MinION platform. The ONT Ligation Sequencing Kit 1D (SQK-LSK108) was applied for the library preparation using the recommended oligo(dT) primers, or custom-made random oligonucleotides, as well as the SuperScipt IV enzyme for the RTs. The cDNA samples were subjected to PCR reactions with KAPA HiFi DNA Polymerase (Kapa Biosystems) and Ligation Sequencing Kit Primer Mix (part of the 1D Kit). The NEBNext End repair/dA-tailing Module (New England Biolabs) was used for the end repair, whereas the NEB Blunt/TA Ligase Master Mix (New England Biolabs) was utilized for the adapter ligation. The enzymatic steps (e.g.: RT, PCR, and ligation) were carried out in a Veriti Cycler (Applied Biosystems) according to the 1D protocol (Moldován et al., 2018b (link); Tombácz et al., 2018b (link)). The Agencourt AMPureXP system (Beckman Coulter) was used for the purification of samples after each enzymatic reaction. The quantity of the libraries was checked using the Qubit Fluorometer 2.0 and the Qubit (ds)DNAHS Assay Kit (Life Technologies). The samples were run on R9.4 SpotON Flow Cells from ONT.

For microbiome analysis, the collected CVF samples were subjected to bacterial DNA extraction using the NucleoSpin Tissue Kit (Macherey–Nagel, Düren, Germany), following the manufacturer’s instructions. Sequencing of 16S rRNA was performed according to the 16S metagenomic sequencing library preparation protocol, targeting the V3 and V4 hypervariable regions. For PCR and purification of the PCR product, the KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, United States) and Agencourt AMPure XP system (Beckman Coulter Genomics, Brea, United States) were used. The initial PCR was performed with 12 ng template DNA using region-specific primers (Supplementary Table 1). After magnetic bead-based purification, a second PCR was performed using primers from the Nextera XT Index Kit (Illumina). Purified PCR products were visualized by gel electrophoresis and quantified using DropSense96 (Trinean, Gentbrugge, Belgium). For quality analysis, the pooled samples were run on an Agilent 2,100 Bioanalyzer (Agilent, Santa Clara, CA, United States). Using the CFX96 Real-Time System, Libraries were quantified by qPCR. After normalization, sequencing of the prepared library was conducted using the MiSeq system (Illumina, San Diego, CA, United States) with 300 bp paired-end reads.

Genomic DNA was extracted from clinical samples from using the QIAmp DNA Blood Mini Kit (Qiagen, Germany) [31 (link),32 (link)]. We performed whole genome amplification (WGA) on the clinical samples using the Illustra GenomiPhi V2 DNA Amplification Kit (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) according to the manufacturer’s instructions. For each WGA reaction, 1 μl of each clinical sample was used, yielding 40 ul of amplified material. Following WGA, we purified the DNA using Agencourt AMPure XP system (Beckman Coulter, Inc., Beverley, MA, USA) according to manufacturer’s instructions. Following genome amplification, we quantified the concentration of total DNA in clinical samples based on OD₂₆₀ using a NanoDrop 3300 Fluorospectrometer (Thermo Scientific, Waltham, MA, USA). Herein, we refer to the DNA concentration as total DNA, for clinical samples since it contains the presence of both human and P. vivax material. All DNA solutions were diluted in 1X Tris-EDTA (TE) Buffer (VWR, Radnor, PA, USA).

Total RNA was extracted from the stored RNAprotect solution using an RNAeasy Qiagen kit (Qiagen, Valencia, CA) as indicated by the manufacturer’s instructions, followed by an additional treatment with DNase I (Qiagen, Valencia, CA) to remove traces of DNA. rRNA depletion and mRNA enrichment were performed using an Illumina Ribo-Zero rRNA removal kit (catalog no. MRZ116C) according to the instructions of the manufacturer (Illumina, San Diego, CA). RNA concentrations were measured using an RNA Qubit assay (Invitrogen, Burlington, Ontario, Canada), and RNA integrity was evaluated using a Bioanalyzer RNA Pico assay (Agilent Technologies, Santa Clara, CA) (18 (link)). cDNA libraries were constructed using a ScriptSeq Complete kit for bacteria (Epicentre, Madison, WI). cDNA was then purified using an Agencourt AMPure XP system (Beckman Coulter, Beverly, MA), and the second cDNA was generated by adding the Illumina adapters as the forward primer and a ScriptSeq index primer as the reverse primer. The resulting libraries were purified using an AMPure XP system (Beckman Coulter, Beverly, MA), quantified with the DNA Qubit assay, and evaluated using the Bioanalyzer sensitivity DNA assay (Agilent Technologies, Santa Clara, CA). One hundred single reads were generated using the Illumina HiSeq 2000 platform at the National Center for Genome Research in Santa Fe, NM.

The 16S primers used in this study are shown in Supplemental Table 2. Six sets of primers for short amplicon sequencing were designed for 16S rDNA variable regions (V2, V3, V4, V6–7, V8, and V9), for 2D sequencing. The pooled PCR product was subjected to 2D DNA library preparation. Primers S-D-Bact-0008-c-S-20 and S-D-Bact-1391-a-A-17, which cover nearly the full length of 16S rDNA, were used for 1D sequencing^{13 (link)–15 (link)}. One nanogram of DNA was used for PCR. KAPA HiFi HotStart ReadyMix (KAPA Biosystems, MA, USA) and Agencourt AMPure XP system (Beckman Coulter Genomics, CA, USA) were used for PCR and PCR product purification, respectively. The PCR conditions are shown in Supplemental Table 3.