title: Metagenomic detection and whole-genome assembly of human pegivirus 2 (HPgV-2) from human plasma samples
design: 14 aliquotted plasma samples from 13 individuals infected with human pegivirus 2 (HPgV-2) were sequenced across 15 sequencing libraries/runs.  For one individual (UC0125.US), three sequencing runs were performed from 2 plasma aliquots.  Plasma samples were clarified of particulate matter by centrifugation at 2650g for 5 min at room temperature, and then were pre-treated with benzonase (500 U/ml; (Sigma)) for 3 hrs at 37oC in 1X benzonase buffer (20 mM Tris-Cl pH 7.5, and 10 mM NaCl, 2 mM MgCl2) to degrade free DNA and RNA. For extractions, the QiaAMP Viral Mini (Qiagen), MagMax Viral RNA (Ambion), and Abbott RNA or total nucleic acid sample preps on the m2000 instrument were performed according to the manufacturer's instructions, all of which successfully recovered HPgV-2. RNA was concentrated using RNA Clean & Concentrator-5 columns (Zymo Research) and eluted in 7 μl of DNase/RNase-Free water.  Randomly primed cDNA libraries were constructed from 5 µl of RNA using the Ovation® RNA-SeqV2 kit (NuGen) according to manufacturer's recommendations. cDNA concentrations were measured on a Qubit Fluorometer using dsDNA BR reagents (Molecular Probes/Life Technologies) and diluted to 0.2 ng/µl. One nanogram (5 µl) was used as input for Nextera XT tagmentation (Illumina) according to manufacturer's instructions, and barcoded libraries were amplified with 16 cycles of PCR. Libraries were quantified on a BioAnalyzer 2200 TapeStation, pooled to a final concentration of 6-12 pM in HT buffer with 1% PhiX loading control, and sequenced on an Illumina MiSeq as 150 nt paired-ends reads using v2 chemistry. To exclude reagent contamination, additional experiments were performed with the SMARTerTM PCR cDNA synthesis (Clontech), Ovation® Single Cell RNA-seq, and Ovation® Human Blood RNA-seq library systems, each of which also generated HPgV-2 sequence.  Barcodes were de-multiplexed on the MiSeq instrument and reads were filtered for Q-scores above 30. FASTQ files were imported into CLC Genomics Workbench 8.0 software (CLC bio/Qiagen, Aarhus, Denmark), Illumina paired-end reads 1 and 2 were merged (paired-end distance 100-250 nt), and duplicate reads were removed. Reads were trimmed for quality (limit=0.05) and ambiguity (2 nt max), with reads below 50 nt discarded; paired-end reads and broken pairs were then aligned to the UC0125.US sequence (Fig. 1E). The following alignment settings were applied: mismatch=2, insertion=3, deletion=3, length fraction=0.7, and similarity fraction=0.8.   Where required, gaps in NGS sequence were filled using One-Step RT-PCR (Qiagen) and Sanger sequenced on an ABI3130 instrument.  Sequences of primers used to characterize the index case and fill in additional genomes are available upon request.  Contigs from multiple runs of NGS or Sanger data were assembled in Sequencher 5.2.3 software to generate strain consensus sequences, and the polyprotein open reading frame was confirmed in DNAStar SeqBuilder software. Finishing the 5' and 3' ends of the HPgV-2 was performed using the First Choice RLM RACE kit (Ambion) according to the manufacturer's instruction.
identifiers.namespace: University of California, San Francisco
identifiers.id: UC0125.US.run3