Computational protocol: Spike Protein Fusion Peptide and Feline Coronavirus Virulence

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Protocol publication

[…] Two 96-well plates of degenerate primers () were designed from aligned reference genomes by using a computational PCR primer design pipeline. The pipeline was developed at the J. Craig Venter Institute (JCVI) to produce tiled amplicons with an optimal length of 550 bp, with 100-bp overlap to provide 6-fold sequence coverage of the genome. An M13 sequence tag was added to the 5′ end of each degenerate primer and was used for sequencing. Primers were arranged in a 96-well plate format, and all PCRs for each sample were performed in 2 plates. The primers used in this study are listed in the .Sequencing reactions were performed by using Big Dye Terminator (Applied Biosystems, Foster City, CA, USA) chemistry. Each amplicon was sequenced from both ends by using M13 primers, and sequencing reactions were analyzed by using a 3730 ABI sequencer (Applied Biosystems). Raw sequence data were trimmed to remove any primer-derived and low-quality sequence; gene sequences were assembled by using a viral assembly tool (www.jcvi.org/cms/research/software). Assemblies were edited computationally and manually. When insufficient underlying sequence information was obtained, the sample was entered into the secondary sequencing pipeline and reamplified by using existing primers or primers designed from the problematic sequence assembly itself. The reamplified sample was then sequenced again.An RNA virus genome prediction program called VIGOR (Viral Genome ORF Reader, JCVI (www.jcvi.org/vigor) can decode many classes of viruses, taking into account virus-specific features, such as alternative splicing, internal open reading frames, and ribosomal slippage. This program was used to annotate de novo assemblies of coronaviruses sequenced at JCVI and also to validate newly assembled genomes during the finishing process. Last, we performed a quality control assessment and manually inspected the gene predictions before loading them into the annotation database at JCVI, from which they were exported in formats acceptable to the National Center for Biotechnology Information. [...] The full-length and partial FCoV genomic nucleotide sequences we obtained were deposited in the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov). The sequence accession numbers of the full-length FCoV sequences used in this study are listed in . The GenBank accession numbers for the partial S gene sequences are JQ304323–JQ304518. Multiple-sequence alignments were constructed by using Clustal W (www.ebi.ac.uk/clustalw) with the Lasergene MegAlign (DNASTAR, www.dnastar.com/t-sub-products-lasergene-megalign.aspx) and MEGA4 (www.megasoftware.net) software programs. To identify key differences between FIPV and FECV, we analyzed their genomes and proteomes; for each nucleotide or amino acid position, we determined the rate at which FIPVs differed from all FECVs at that position. Phylogenetic analysis was performed by using features of the MEGA4 suite of programs. Phylogenetic trees of these sequences were obtained by using the neighbor-joining method. The bootstrap consensus tree, inferred from 1,000 replicates, was prepared; positions containing gaps and missing data were eliminated from the dataset. […]

Pipeline specifications

Software tools VIGOR, Clustal W, MEGA
Applications Phylogenetics, WGS analysis
Organisms Feline coronavirus, Feline infectious peritonitis virus
Diseases Feline Infectious Peritonitis, Severe Acute Respiratory Syndrome