Genome sequencing of Lone Star virus, a novel bunyavirus in the Amblyomma americanum tick
LSV-infected Vero cell cultures were extracted using two different protocols, one to purify RNA from viral particles (“virus purification protocol”) and the other to extract total RNA.  First, 130 microL of viral culture supernatant was treated with Turbo DNase, Baseline Zero DNase, Benzonase, and RNase A (Roche, South San Francisco, CA), and then extracted with the QIAamp UltraSens Virus Kit (Qiagen, Valencia, CA).  A second RNA extraction without pre-nuclease treatment was performed on 400 micorL of supernatant using TRIzol reagent (Life Technologies, Foster City, CA).  Each extraction was performed according to the manufacturer’s protocol.  An LSV cDNA library for Illumina sequencing was prepared with the ScriptSeq version 2 kit according to the manufacturer’s protocol (Epicentre, Madison, WI).  The input consisted of 30 ng of RNA from the first extraction and 80 ng from the second extraction.  RNA was reverse-transcribed to cDNA, adaptors were ligated to the cDNA ends, and the cDNA was then amplified with 17 cycles of PCR.  AMPure XP beads (Beckman Coulter Genomics, Brea, CA) were used to remove primer and adaptor dimers as well as larger PCR fragments ( more than 600 bp) from the amplified cDNA library.  Library size distribution and concentration were determined using a High Sensitivity DNA kit on an Agilent Bioanalyzer 2100 instrument (Agilent, Santa Clara, CA) and a KAPA Library Quantification Kit (Kapa Biosystems, Woburn, MA), respectively.  Approximately 10 pM of library was used for 150-bp paired-end sequencing on an Illumina MiSeq Sequencer (Illumina, Hayward, CA).
University of California, San Francisco
LSV