Clc genomic workbench v7.5

The genomic DNA from strain 863 was extracted using the Masterpure DNA Purification Kit (Epicentre, Madison, WI, USA) according to the manufacturer’s protocol. The quality of the genomic DNA was assessed using a Nanodrop Spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). The extracted DNA was used in Nextera Library Preparation Kit (Illumina San Diego, CA, USA). The prepared library was sequenced using 100 bp × 2 cartridge in HiSeq 2500 High Throughput Sequencer (Illumina) on rapid run mode. The quality of the sequenced data was assessed using FastQC software [22 ]. The sequenced data were then trimmed and assembled using CLC Genomic Workbench (V7.5; Qiagen, Hilden, Germany). Following this, the assembled sequence was annotated by the National Center for Biotechnology Information (NCBI) Prokaryotic Genome Automatic Annotation Pipeline (PGAAP) [23 (link)] and RAST [24 (link)].

The modules of EditSeq and MegAlign of DNASTAR Lasergene 12 Core Suite (DNASTAR, Inc. Madison, WI, USA) were used for viral open reading frames (ORFs) prediction, amino acid (aa) translation, sequence alignment, and pair-wised sequence comparison. An online search program (http://blast.ncbi.nlm.nih.gov/Blast.cgi) identified the highest similarities between the studied ARV genome segment and the published sequences.
Sequencing coverage, mapped reads, and intra-host single-nucleotide variants (iSNVs) of each assembled contigs were calculated and visualized by CLC Genomic Workbench V7.5 software (QIAGEN, Boston, MA, USA). Phylogenetic analysis of genome segments were carried out by using the neighbor-joining method in MEGA CC program [41 (link)] and the bootstrap validation method with 1000 replications. The visualization of genome alignment was performed using the mVISTA (http://genome.lbl.gov/vista/mvista/submit.shtml) and the scale sequence was using studied pheasant-origin ARV genome. Genome sequences of 13 ARV reference strains were retrieved from Genbank for sequence comparisons (S1 Table).