Genome Sequencing, Assembly, and Annotation

DNA concentration and purity were checked by a spectrophotometer (Nanodrop 2000, Thermo Scientific, Waltham, MA, United States), and the DNA integrity was analyzed by agarose gel electrophoresis. Then, the genomic DNA was sent to Tianjin Biochip Corporation (Tianjin, China), a commercial NGS service provider, for whole genome sequencing. The complete genome of GLB197 was sequenced by the Illumina HiSeq and Pacific Biosciences (PacBio, Menlo Park, CA, United States) platforms. Briefly, genomic DNA sample were fragmented using an ultrasonication approach (Covaris, Woburn, MA, United States) according to the manufacturer’s instruction. The 10-kb template library was constructed using DNA Template Prep Kit 2.0 with the 10-kb insert library protocol. The library was sequenced using the PacBio RSII Sequencer. Meantime, 1 μg DNA sample was also fragmented using an ultrasonication approach, size selected and end repaired. Each generated fragment was ligated to Illumina-specific adapter sequence, quantified, indexed, and sequenced on the Hiseq platform of Illumina. Quality-of-sequence reads were first analyzed using FastQC tool. Then, adaptor sequence removal, trimming, error correction, and assembly were performed using the HGAP software (Chin et al., 2013). Gene predictions were performed with Prokka version 1.11 which predicts coding DNA sequence (CDS) using Prodigal (Seemann, 2014). Annotation of the protein-coding sequence was conducted using the Basic Local Alignment Search Tool (BLAST) against the COG, Kyoto Encyclopedia of Genes and Genomes, and Interpro databases. The final annotated chromosome was plotted using CIRCOS to show the gene locations, GC skew, and GC content (Krzywinski et al., 2009).