Computational protocol: Complete sequence determination of a novel reptile iridovirus isolated from soft-shelled turtle and evolutionary analysis of Iridoviridae

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Protocol publication

[…] Sequencing of STIV genome was carried out using a pyrosequencing platform, the Genome Sequencer 20 (GS20) System (454 Life Science Corporation, Roche). Briefly, after the quality of STIV genome DNA had been assessed by agarose gel electrophoresis and analysed by Agilent bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), ~10 μg samples were sheared by nebulization into 300–500 bp fragments. The whole genomic library was amplified using GS20 emPCR kits and sequenced with the 454 Life Science GS 20 instrument according to the manufacturer's recommendations. The GS De Novo Assembler software generates a consensus sequence of the whole DNA sample by assembly of de novo shotgun sequencing reads into contigs and subsequent ordering of these contigs into scaffolds. The average reading frame length was about 100 bp with 20-fold coverage of the whole genome. To fill the gaps, 16 oligonucleotide primers were used to amplify by polymerase chain reaction (PCR) directly from the genome DNA and the corresponding PCR products were sequenced using an automated ABI 3730 apparatus (Applied Biosystems, Shanghai, China). [...] Nucleotide and amino acid sequences were analyzed using the DNASTAR software package (Lasergene, Madison, WI, USA). The genomic organization was drawn using the DNAMAN program. Nucleotide sequence and protein database searches were performed using the BLAST programs at the NCBI website . The whole genome sequence was also submitted to (Softberry Inc., Mount Kisco, NY, USA) for identification of all putative ORFs. For more refined analyses, conserved motifs and domains and putative functions of deduced STIV proteins composed of 40 or more amino acids with homologies to other proteins in sequence databases were identified using several online programs as follows: for conserved motifs and domains, and were used; for transmembrane domain predictions, was used. DNA repetitive sequences were detected computationally using REPuter and a tandem repeats finder []. The STIV microRNA prediction was carried out as described []. [...] To analyze the evolutionary position of STIV in the family Iridoviridae, four conserved iridovirus genes, which are also present in other large DNA viruses, were evaluated using the PHYLIP program based on the amino acid alignment. Multiple alignments of proteins and nucleotide sequences were generated using the MAFFT 6 and ClustalX programs [,]. In addition, a phylogenetic tree was constructed using MEGA version 4 with complete genomic sequences corresponding to the available sequencing data of iridoviruses. [...] All the putative iridovirus genes were obtained from NCBI databases and the all-against-all BLASTP similarity search was performed. The different iridovirus genes were regarded as COGs based on protein sequence similarity. The homologs were determined if one hit the other in the BLASTP search with an e-value ≤ 10-5 and the maximal produced alignments covered at least 60% of the longer protein, while the homologous proteins from multiple copies of a gene in one genome were counted only once. Eleven sets of COGs were aligned independently using the ClustalX alignment program, then the alignments were concatenated into a single alignment and a neighbor-joining (NJ) tree was constructed using MEGA version 4. Gene gain and loss events were processed with PAML software package and assigned to branches in the phylogenetic tree []. […]

Pipeline specifications

Software tools Newbler, DNAMAN, REPuter, TRF, PHYLIP, MAFFT, Clustal W, MEGA, BLASTP, PAML
Applications Phylogenetics, WGS analysis
Organisms Pelodiscus sinensis, Frog virus 3, Hoplobatrachus tigerinus, Singapore grouper iridovirus, Great Island virus
Diseases HIV Infections, Mastocytosis, Systemic